G-type peptides to ameliorate atherosclerosis

ABSTRACT

This invention provides novel peptides that ameliorate one or more symptoms of atherosclerosis and/or other pathologies characterized by an inflammatory response. In certain embodiment, the peptides resemble a G* amphipathic helix of apolipoprotein J. The peptides are highly stable and readily administered via an oral route.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0001] This work was supported, in part, by Grant No: HL30568 from theNational Heart Blood Lung Institute of the National Institutes ofHealth. The Government of the United States of America may have certainrights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] [Not Applicable]

FIELD OF THE INVENTION

[0003] This invention relates to the field of atherosclerosis. Inparticular, this invention pertains to the identification of a class ofpeptides that are orally administrable and that ameliorate one or moresymptoms of atherosclerosis or other pathologies characterized by aninflammatory response.

BACKGROUND OF THE INVENTION

[0004] The introduction of statins (e.g. Mevacor@, Lipitor@) has reducedmortality from heart attack and stroke by about one-third. However,heart attack and stroke remain the major cause of death and disability,particularly in the United States and in Western European countries.Heart attack and stroke are the result of a chronic inflammatorycondition, which is called atherosclerosis.

[0005] Several causative factors are implicated in the development ofcardiovascular disease including hereditary predisposition to thedisease, gender, lifestyle factors such as smoking and diet, age,hypertension, and hyperlipidemia, including hypercholesterolemia.Several of these factors, particularly hyperlipidemia andhypercholesteremia (high blood cholesterol concentrations) provide asignificant risk factor associated with atherosclerosis.

[0006] Cholesterol is present in the blood as free and esterifiedcholesterol within lipoprotein particles, commonly known aschylomicrons, very low density lipoproteins (VLDLs), low densitylipoproteins (LDLs), and high density lipoproteins (HDLs). Concentrationof total cholesterol in the blood is influenced by (1) absorption ofcholesterol from the digestive tract, (2) synthesis of cholesterol fromdietary constituents such as carbohydrates, proteins, fats and ethanol,and (3) removal of cholesterol from blood by tissues, especially theliver, and subsequent conversion of the cholesterol to bile acids,steroid hormones, and biliary cholesterol.

[0007] Maintenance of blood cholesterol concentrations is influenced byboth genetic and environmental factors. Genetic factors includeconcentration of rate-limiting enzymes in cholesterol biosynthesis,concentration of receptors for low density lipoproteins in the liver,concentration of rate-limiting enzymes for conversion of cholesterolsbile acids, rates of synthesis and secretion of lipoproteins and genderof person. Environmental factors influencing the hemostasis of bloodcholesterol concentration in humans include dietary composition,incidence of smoking, physical activity, and use of a variety ofpharmaceutical agents. Dietary variables include amount and type of fat(saturated and polyunsaturated fatty acids), amount of cholesterol,amount and type of fiber, and perhaps amounts of vitamins such asvitamin C and D and minerals such as calcium.

[0008] Low density lipoprotein (LDL) oxidation has been stronglyimplicated in the pathogenesis of atherosclerosis. High densitylipoprotein (HDL) has been found to be capable of protecting against LDLoxidation, but in some instances has been found to accelerate LDLoxidation. Important initiating factors in atherosclerosis include theproduction of LDL-derived oxidized phospholipids.

[0009] Normal HDL has the capacity to prevent the formation of theseoxidized phospholipids and also to inactivate these oxidizedphospholipids once they have formed. However, under some circumstancesHDL can be converted from an anti-inflammatory molecule to apro-inflammatory molecule that actually promotes the formation of theseoxidized phospholipids.

[0010] HDL and LDL have been suggested to be part of the innate immunesystem (Navab et al. (2001) Arterioscler Thromb Vasc Biol. 21: 481-488).The generation of anti-inflammatory HDL has been achieved with class Aamphipathic helical peptides that mimic the major protein of HDL,apolipoprotein A-I (apo A-I) (see, e.g., WO 02/15923).

SUMMARY OF THE INVENTION

[0011] This invention provides novel compositions and methods toameliorate symptoms of atherosclerosis and other inflammatory conditionssuch as rheumatoid arthritis, lupus erythematous, polyarteritis nodosa,osteoporosis, Altzheimer's disease and viral illnesses such as influenzaA.

[0012] In certain embodiments this invention provides “isolated”polypeptides that ameliorate a symptom of atherosclerosis or otherpathologies associated with an inflammatory response and/or compositionscomprising such polypeptides. The polypeptides typically comprise anamphipathic helical polypeptide having charged residues on the polarface of the polypeptide and possessing a wide non-polar face. Thepolypeptide is typically at least about 10 amino acids in length and/orabout 40 or fewer polypeptides in length. Preferred polypeptidestypically comprise a G* amphipathic helix. In certain embodiments, thepolypeptides show greater than about 50%, preferably greater than about75%, and more preferably greater than about 85% sequence identity withapo J (e.g. over a domain the same length as the polypeptide inquestion). Preferred polypeptides of this invention protect aphospholipid (e.g.,1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC),1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC)),1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine (SAPE))against oxidation by an oxidizing agent (e.g., 13(S)-HPODE, 15(S)-HPETE,HPODE, HPETE, HODE, and HETE). Particularly preferred polypeptidescomprise or consist of one or more of the following amino acidsequences: LLEQLNEQFNWVSRLANLTEGE, (SEQ ID NO:1), LLEQLNEQFNWVSRLANL,(SEQ ID NO:2), NELQEMSNQGSKYVNKEIQNAVNGV, (SEQ ID NO:3), IQNAVNGVKQIKTLIEKTNEE, (SEQ ID NO:4), RKTLLSNLEEAKKKKEDALNETRESETKLKEL, (SEQ IDNO:5), PGVCNETMMALWEECK, (SEQ ID NO:6), PCLKQTCMKFYARVCR, (SEQ ID NO:7),ECKPCLKQTCMKFYARVCR, (SEQ ID NO:8), LVGRQLEEFL, (SEQ ID NO:9),MNGDRIDSLLEN, (SEQ ID NO:10), QQTHMLDVMQD, (SEQ ID NO:11),FSRASSIIDELFQD, (SEQ ID NO:12), PFLEMIHEAQQAMDI, (SEQ ID NO:13),PTEFIREGDDD, (SEQ ID NO:14), RMKDQCDKCREILSV, (SEQ ID NO:15),PSQAKLRRELDESLQVAERLTRKYNELLKSYQ, (SEQ ID NO:16), LLEQLNEQFNWVSRLANLTQGE, (SEQ ID NO:17), DQYYLRVTTVA, (SEQ ID NO:18),PSGVTEVVVKLFDS, (SEQ ID NO:19), PKFMETVAEKALQEYRKKHRE, (SEQ ID NO:20),WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO:21), VATVMWDYFSQ LSNNAKEAVEHLQK(SEQ ID NO:22), RWELALGRFWDYLRWVQTLSEQVQEEL (SEQ ID NO:23),LSSQVTQELRALMDETMKELKELKAYKSELEEQLT (SEQ ID NO:24),ARLSKELQAAQARLGADMEDVCGRLV (SEQ ID NO:25), VRLASHLRKLRKRLLRD ADDLQKRLA(SEQ ID NO:26), PLVEDMQRQWAGLVEKVQA (SEQ ID NO:27), MSTYTGIFTDQVLSVLK(SEQ ID NO:28), and LLSFMQGYMKHATKTAKDALSS (SEQ ID NO:29). In certainembodiments, the polypeptide is a concatamer of two or more of theseamino acid sequences and/or a concatamer of one or more of these aminoacid sequences and an apo A-I sequence or a mimetic thereof (see, e.g.,PCT publication WO 02/15923 for apo A-I related polypeptides/mimetics).The polypeptides of this invention can comprise a protecting group (e.g.a protecting group on the amino and/or carboxyl terminus). Preferredprotecting groups include, but are not limited to acetyl, amide, 3 to 20carbon alkyl groups, Fmoc, t-boc, 9-fluoreneacetyl group,1-fluorenecarboxylic group, 9-florenecarboxylic group,9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan),Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt),4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl(Mts), 4,4=-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl(MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom),t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum),t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), a benzoyl group, acarbobenzoxy group, a propyl group, a butyl group, a pentyl group, ahexyl group, and Trifluoroacetyl (TFA). In certain embodiments, thepolypeptide comprises a protecting group coupled to the amino terminaland the amino terminal protecting group is a protecting group such as abenzoyl group, an acetyl, a propeonyl, a carbobenzoxy, a propyl, abutyl, a pentyl, a hexyl, or a 3 to 20 carbon alkyl. In certainembodiments, the polypeptide comprises a protecting group coupled to thecarboxyl terminal and the carboxyl terminal protecting group is anamide.

[0013] In particularly preferred embodiments, the polypeptide(s) of thisinvention comprise one or more dextro “D” amino acids. In certainembodiments, the polypeptide(s) of this invention comprise at least two,preferably at least 4, and most preferably all “D” amino acids.

[0014] In certain embodiments the polypeptide(s) described herein arecovalently coupled to a phospholipid (e.g. lysophosphatidyl choline). Inparticularly preferred embodiments, the polypeptide(s) are coupled tothe sn-1 or sn-2 position of a phospholipid (e.g. propionoyl, butanoyl,pentanoyl, caproyl, heptanoyl, capryloyl, nonanoyl, capryl, undcanoyl,lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl,heptadecanoyl, stearoyl, nonadecanoyl, arachidoyl, heniecosanoyl,behenoyl, trucisanoyl, lignoceroyl, myristoleoyl (9-cis), myristelaidoyl(9-trans), palmitoleoyl (9-cis), palmitelaidoyl (9-trans, and the like).

[0015] The polypeptide(s) of this invention can be formulated with apharmacologically acceptable excipient (e.g. a unit dosage formulationfor oral administration, rectal administration, nasal administration,injection, and the like).

[0016] In another embodiment, this invention provides a compositionsuitable for oral administration that ameliorates a symptom ofatherosclerosis or other pathologies characterized by an inflammatoryresponse. The composition comprises a polypeptide comprising anamphipathic helix (e.g. a G* helix) as described herein where thepolypeptide comprises one or more “D” amino acids as described hereinand the polypeptide is blocked at the amino terminus and the carboxylterminus as described herein.

[0017] In certain embodiments, this invention provides pharmaceuticalformulations (compositions). The pharmaceuticals comprise a polypeptideas described herein in a pharmaceutically acceptable excipient. Theformulation is often a unit dosage formulation (e.g. for oral, rectal,nasal, or injectible administration to a mammal such as a human).

[0018] This invention also provides a method of method of ameliorating asymptom of atherosclerosis, or other pathology characterized by aninflammatory response in a mammal. The method involves administering tothe mammal (e.g. a human) a polypeptide or a concatamer of a polypeptidecomprising an amphipathic helical polypeptide having charged residues onthe polar face of the polypeptide and possessing a wide non-polar faceas described herein. In certain embodiments, the mammal is a human (e.g.a human diagnosed as having or as being at risk for atherosclerosis,stroke, or other pathology associated with an inflammatory response). Incertain embodiments, the mammal is non-human mammal (e.g. canine,feline, bovine, equine, porcine, etc.).

[0019] In another embodiment, this invention provides a method ofameliorating a symptom of a pathology characterized by an inflammatoryresponse (e.g. a symptom of rheumatoid arthritis, lupus erythematous,polyarteritis nodosa, osteoporosis, Altzheimer's disease and viralillnesses such as influenza A, etc). The method involves administeringto the mammal (e.g. human) a polypeptide or a concatamer of apolypeptide comprising an amphipathic helical polypeptide having chargedresidues on the polar face of the polypeptide and possessing a widenon-polar face as described herein.

[0020] This invention also provides a kit for ameliorating a symptom ofatherosclerosis or another pathology characterized by an inflammatoryresponse. The kit typically includes a container containing one or moreof the polypeptides described herein. The polypeptide(s) can be combinedwith a pharmaceutically acceptable excipient (e.g. in a unit dosageformulation for oral, nasal, rectal, injectible administration). The kitcan additionally include instructional materials teaching the use of thepolypeptide for ameliorating one or more symptoms of atherosclerosis orof a pathology characterized by an inflammatory response.

[0021] In still another embodiment, this invention provides a method ofmitigating or preventing a coronary complication associated with anacute phase response to an inflammation in a mammal, wherein saidcoronary complication is a symptom of atherosclerosis. The methodinvolves administering to a mammal having the acute phase response, orat risk for the acute phase response, one or more polypeptides describedherein. The administration can be by a route such as oraladministration, nasal administration, rectal administration,intraperitoneal injection, and intravascular injection, subcutaneousinjection, transcutaneous administration, intramuscular injection, andthe like. In certain embodiments, the polypeptide is administered incombination with an all L-form of the same polypeptide. In certainembodiments, the polypeptide(s) are provided as a unit formulation in apharmaceutically acceptable excipient. The acute phase response can bean inflammatory response associated with a recurrent inflammatorydisease. In certain embodiments, the acute phase response is associatedwith a disease including, but not limited to leprosy, tuberculosis,systemic lupus erythematosus, polymyalgia rheumatica, polyarteritisnodosa, scleroderma, idiopathic pulmonary fibrosis, chronic obstructivepulmonary disease, Alzheimers Disease and AIDS, polymyalgia rheumatica,polyarteritis nodosa, scleroderma, idiopathic pulmonary fibrosis,chronic obstructive pulmonary disease, Alzheimers Disease, AIDS,coronary calcification, calcific aortic stenosis, osteoporosis, andrheumatoid arthritis. In certain embodiments, the acute phase responseis an inflammatory response associated with a condition such as abacterial infection, a viral infection, a fungal infection, an organtransplant, a wound, an implanted prosthesis, parasitic infection,sepsis, endotoxic shock syndrome, and biofilm formation.

[0022] This invention also provides a method of mitigating or preventinga coronary complication associated with an acute phase response to aninflammation in a mammal where the coronary complication is a symptom ofatherosclerosis. The method involves assaying the mammal (e.g. a human)for an acute phase protein (APP) level indicative of an acute phaseresponse or a significant risk of an acute phase response; andadministering to a mammal showing an acute phase protein (APP) levelindicative of an acute phase response a polypeptide as described herein.The acute phase protein (APP) can be a positive APR such as serumamyloid A, c-reactive protein, serum amyloid P component, C2 complementprotein, C3 complement protein, C4 complement protein, C5 complementprotein, C9 complement protein, B complement protein, C1 inhibitor, C4binding protein, fibrinogen, von Willebrand factor, α1-antitrypsin,α1-antichymotrypsin, α2 antiplasmin, heparin cofactor II, plasminogenactivator inhibitor I, haptoglobin, haemopexin, ceruloplasmin, manganesesuperoxide dismutase, (α1-acid glycoprotein, haeme oxygenase, mannosebinding protein, leukocyte protein I, lipoprotein (a), andlipopolysaccharide binding protein and/or a negative APR such asconsisting of albumin, prealbumin, transferin, apoAI, apoAII, α2-HSglycoprotein, inter-α-trypsin inhibitor, histidine rich glycoprotein.

[0023] Definitions.

[0024] The terms “isolated”, “purified”, or “biologically pure” whenreferring to an isolated polypeptide refer to material that issubstantially or essentially free from components that normallyaccompany it as found in its native state. With respect to nucleic acidsand/or polypeptides the term can refer to nucleic acids or polypeptidesthat are no longer flanked by the sequences typically flanking them innature. Chemically synthesized polypeptides are “isolated” because theyare not found in a native state (e.g. in blood, serum, etc.). In certainembodiments, the term “isolated” indicates that the polypeptide is notfound in nature.

[0025] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

[0026] The term “an amphipathic helical peptide” refers to a peptidecomprising at least one amphipathic helix (amphipathic helical domain).Certain amphipathic helical peptides of this invention can comprise twoor more (e.g. 3, 4, 5, etc.) amphipathic helices.

[0027] The term “class A amphipathic helix” refers to a proteinstructure that forms an α-helix producing a segregation of a polar andnonpolar faces with the positively charged residues residing at thepolar-nonpolar interface and the negatively charged residues residing atthe center of the polar face (see, e.g., “Segrest et al. (1990)Proteins: Structure, Function, and Genetics 8:103-117).

[0028] “Apolipoprotein J” (apo J) is known by a variety of namesincluding clusterin, TRPM2, GP80, and SP 40,40 (Fritz (1995) Pp 112 In:Clusterin: Role in Vertebrate Development, Function, and Adaptation(Harmony JAK Ed.), R. G. Landes, Georgetown, Tex.,). It was firstdescribed as a heterodimeric glycoprotein and a component of thesecreted proteins of cultured rat Sertoli cells (Kissinger et al. (1982)Biol Reprod; 27:233240). The translated product is a single-chainprecursor protein that undergoes intracellular cleavage into adisulfide-linked 34 kDa αsubunit and a 47 kDa βsubunit Collard andGriswold (187) Biochem., 26: 3297-3303). It has been associated withcellular injury, lipid transport, apoptosis and it may be involved inclearance of cellular debris caused by cell injury or death. Clusterinhas been shown to bind to a variety of molecules with high affinityincluding lipids, peptides, and proteins and the hydrophobic probe1-anilino-8-naphthalenesulfonate (Bailey et al. (2001) Biochem., 40:11828-11840).

[0029] The class G amphipathic helix is found in globular proteins, andthus, the name class G. The feature of this class of amphipathic helixis that it possesses a random distribution of positively charged andnegatively charged residues on the polar face with a narrow nonpolarface. Because of the narrow nonpolar face this class does not readilyassociate with phospholipid (see, Segrest et al. (1990) Proteins:Structure, Function, and Genetics. 8: 103-117; also see Erratum (1991)Proteins: Structure, Function and Genetics, 9: 79). Several exchangeableapolipoproteins possess similar but not identical characteristics to theG amphipathic helix. Similar to the class G amphipathic helix, thisother class possesses a random distribution of positively and negativelycharged residues on the polar face. However, in contrast to the class Gamphipathic helix which has a narrow nonpolar face, this class has awide nonpolar face that allows this class to readily bind phospholipidand the class is termed G* to differentiate it from the G class ofamphipathic helix (see Segrest et al. (1992) J. Lipid Res., 33: 141-166;also see Anantharamaiah et al. (1993) Pp. 109-142 In: The AmphipathicHelix, Epand, R. M. Ed CRC Press, Boca Raton, Fla.). Computer programsto identify and classify amphipathic helical domains have been describedby Jones et al.(1992) J. Lipid Res. 33: 287-296) and include, but arenot limited to the helical wheel program (WHEEL or WHEEL/SNORKEL),helical net program (HELNET, HELNET/SNORKEL, HELNET/Angle), program foraddition of helical wheels (COMBO or COMBO/SNORKEL), program foraddition of helical nets (COMNET, COMNET/SNORKEL, COMBO/SELECT,COMBO/NET), consensus wheel program (CONSENSUS, CONSENSUS/SNORKEL), andthe like.

[0030] The term “ameliorating” when used with respect to “amelioratingone or more symptoms of atherosclerosis” refers to a reduction,prevention, or elimination of one or more symptoms characteristic ofatherosclerosis and/or associated pathologies. Such a reductionincludes, but is not limited to a reduction or elimination of oxidizedphospholipids, a reduction in atherosclerotic plaque formation andrupture, a reduction in clinical events such as heart attack, angina, orstroke, a decrease in hypertension, a decrease in inflammatory proteinbiosynthesis, reduction in plasma cholesterol, and the like.

[0031] The term “enantiomeric amino acids” refers to amino acids thatcan exist in at least two forms that are nonsuperimposable mirror imagesof each other. Most amino acids (except glycine) are enantiomeric andexist in a so-called L-form (L amino acid) or D-form (D amino acid).Most naturally occurring amino acids are “L” amino acids. The terms “Damino acid” and “L amino acid” are used to refer to absoluteconfiguration of the amino acid, rather than a particular direction ofrotation of plane-polarized light. The usage herein is consistent withstandard usage by those of skill in the art.

[0032] The term “protecting group” refers to a chemical group that, whenattached to a functional group in an amino acid (e.g. a side chain, analpha amino group, an alpha carboxyl group, etc.) blocks or masks theproperties of that functional group. Preferred amino-terminal protectinggroups include, but are not limited to acetyl, or amino groups. Otheramino-terminal protecting groups include, but are not limited to alkylchains as in fatty acids, propeonyl, formyl and others. Preferredcarboxyl terminal protecting groups include, but are not limited togroups that form amides or esters.

[0033] The phrase “protect a phospholipid from oxidation by an oxidizingagent” refers to the ability of a compound to reduce the rate ofoxidation of a phospholipid (or the amount of oxidized phospholipidproduced) when that phospholipid is contacted with an oxidizing agent(e.g. hydrogen peroxide, 13-(S)-HPODE, 15-(S)-HPETE, HPODE, HPETE, HODE,HETE, etc.).

[0034] The terms “low density lipoprotein” or “LDL” is defined inaccordance with common usage of those of skill in the art. Generally,LDL refers to the lipid-protein complex which when isolated byultracentrifugation is found in the density range d=1.019 to d=1.063.

[0035] The terms “high density lipoprotein” or “HDL” is defined inaccordance with common usage of those of skill in the art. Generally“HDL” refers to a lipid-protein complex which when isolated byultracentrifugation is found in the density range of d=1.063 to d=1.21.

[0036] The term “Group I HDL” refers to a high density lipoprotein orcomponents thereof (e.g. apo A-I, paraoxonase, platelet activatingfactor acetylhydrolase, etc.) that reduce oxidized lipids (e.g. in lowdensity lipoproteins) or that protect oxidized lipids from oxidation byoxidizing agents.

[0037] The term “Group II HDL” refers to an HDL that offers reducedactivity or no activity in protecting lipids from oxidation or inrepairing (e.g. reducing) oxidized lipids.

[0038] The term “HDL component” refers to a component (e.g. molecules)that comprises a high density lipoprotein (HDL). Assays for HDL thatprotect lipids from oxidation or that repair (e.g. reduce oxidizedlipids) also include assays for components of HDL (e.g. apo A-I,paraoxonase, platelet activating factor acetylhydrolase, etc.) thatdisplay such activity.

[0039] The term “human apo A-I peptide” refers to a full-length humanapo A-I peptide or to a fragment or domain thereof comprising a class Aamphipathic helix.

[0040] A “monocytic reaction” as used herein refers to monocyte activitycharacteristic of the “inflammatory response” associated withatherosclerotic plaque formation. The monocytic reaction ischaracterized by monocyte adhesion to cells of the vascular wall (e.g.cells of the vascular endothelium), and/or chemotaxis into thesubendothelial space, and/or differentiation of monocytes intomacrophages.

[0041] The term “absence of change” when referring to the amount ofoxidized phospholipid refers to the lack of a detectable change, morepreferably the lack of a statistically significant change (e.g. at leastat the 85%, preferably at least at the 90%, more preferably at least atthe 95%, and most preferably at least at the 98% or 99% confidencelevel). The absence of a detectable change can also refer to assays inwhich oxidized phospholipid level changes, but not as much as in theabsence of the protein(s) described herein or with reference to otherpositive or negative controls.

[0042] The following abbreviations are used herein: PAPC:L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC:1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine; PGPC:1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC:1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phosphocholine;ChC18:2: cholesteryl linoleate; ChC18:2-OOH: cholesteryl linoleatehydroperoxide; DMPC: 1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine;PON: paraoxonase; HPF: Standardized high power field; PAPC:L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; BL/6:C57BL/6J; C3H:C3H/HeJ.

[0043] The term “conservative substitution” is used in reference toproteins or peptides to reflect amino acid substitutions that do notsubstantially alter the activity (specificity (e.g. for lipoproteins))orbinding affinity (e.g. for lipids or lipoproteins)) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0044] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.With respect to the peptides of this invention sequence identity isdetermined over the full length of the peptide.

[0045] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

[0046] Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

[0047] One example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle(1987) J. Mol. Evol. 35:351-360. The method used is similar to themethod described by Higgins & Sharp (1989) CABIOS 5: 151-153. Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

[0048] Another example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described in Altschul et al. (1990) J. Mol. Biol.215: 403-410. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0049] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad.Sci. USA, 90: 5873-5787). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIG. 1 shows a comparison of the effect of D4F (Navab, et al.(2002) Circulation, 105: 290-292) and apoj peptide 336 made from D aminoacids (D-J336*) on the prevention of LDL-induced monocyte chemotacticactivity in vitro in a co-incubation experiment. The data are mean±SD ofthe number of migrated monocytes in nine high power fields in quadruplecultures. (D-J336=Ac-LLEQLNEQFNWVSRLANLTEGE-NH₂, SEQ ID NO:1).

[0051]FIG. 2 illustrates the prevention of LDL-induced monocytechemotactic activity by pre-treatment of artery wall cells with D-J336as compared to D-4F. The data are mean±SD of the number of migratedmonocytes in nine high power fields in quadruple cultures.

[0052]FIG. 3 illustrates he effect of apo J peptide mimetics on HDLprotective capacity in LDL receptor null mice. The values are themean±SD of the number of migrated monocytes in 9 high power fields fromeach of quadruple assay wells.

[0053]FIG. 4 illustrates protection against LDL-induced monocytechemotactic activity by HDL from apo E null mice given oral peptides.The values are the mean±SD of the number of migrated monocytes in 9 highpower fields from each of quadruple assay wells. Asterisks indicatesignificant difference (p<0.05) as compared to No Peptide mHDL.

[0054]FIG. 5 illustrates the effect of oral apo A-I peptide mimetic andapoj peptide on LDL susceptibility to oxidation. The values are themean±SD of the number of migrated monocytes in 9 high power fields fromeach of quadruple assay wells. Asterisks indicate significant difference(p<0.05) as compared to No Peptide LDL.

[0055]FIG. 6 illustrates the effect of oral apoA-1 peptide mimetic andapoJ peptide on HDL protective capacity. The values are the mean±SD ofthe number of migrated monocytes in 9 high power fields from each ofquadruple assay wells. Asterisks indicate significant difference(p<0.05) as compared to No Peptide mHDL.

[0056]FIG. 7 illustrates the Effect of oral apoA-1 peptide mimetic andapoj peptide on plasma paraoxonase activity. The values are the mean±SDof readings from quadruple plasma aliquots. Asterisks indicatesignificant differences (p<0.05) as compared to No Peptide controlplasma.

DETAILED DESCRIPTION

[0057] I. Mitigation of a Symptom of Atherosclerosis.

[0058] This invention pertains to the surprising discovery thatamphipathic helical peptide analogues possessing distributed (e.g.randomly distributed, haphazardly distributed, etc.) charged residues onthe polar face of the peptide possess anti-inflammatory properties andare capable of mediating a symptom of atherosclerosis or other pathologycharacterized by an inflammatory response (e.g., rheumatoid arthritis,lupus erythematous, polyarteritis nodosa, and osteoporosis). Preferredpeptides of this invention generally mimic the amphipathic helicaldomain(s) of apolipoprotein J (apo J).

[0059] In certain preferred embodiments, the peptides are amphipathichelical peptide analogues possessing distributed charged residues(positively and/or negatively charged residues) on the polar face of thepeptide and possessing a wide nonpolar face (termed a globular proteinlike, G*) amphipathic helical domain. Such amphipathic helical G*domains are characteristic of apo J and certain other apoproteins (e.g.apo AI, apo AIV, apo E, apo CII, apo CIII, and the like, but not apoA-II or apo C-I). The peptides of this invention preferably range fromabout 10 to about 100 amino acids in length, more preferably from about10 to about 60 or 80 amino acids in length, and most preferably fromabout 10, 15, or 20 amino acids to about 40 or 50 amino acids in length.In certain embodiments, the peptides range from about 10 to about 40amino acids in length. Certain particularly preferred peptides of thisinvention show greater than about 40%, preferably greater than about 50%or 60%, more preferably greater than about 70% or 80% and mostpreferably greater than about 90% or 95% sequence identity with apo J orfragments thereof (ranging in length from about 10 to about 40 aminoacids, e.g. over the same length as the peptide in question).

[0060] It was a surprising discovery of this invention that suchpeptides, particularly when comprising one or more D-form amino acidsretain the biological activity of the corresponding L-form peptide.Moreover, these peptides show in vivo activity, even when deliveredorally. The peptides show elevated serum half-life, and the ability tomitigate or prevent/inhibit one or more symptoms of atherosclerosis.

[0061] We discovered that normal HDL inhibits three steps in theformation of mildly oxidized LDL. In those studies (see, e.g. WO02/15923) we demonstrated that treating human LDL in vitro with apo A-Ior an apo A-I mimetic peptide (37 pA) removed seeding molecules from theLDL that included HPODE and HPETE. These seeding molecules were requiredfor cocultures of human artery wall cells to be able to oxidize LDL andfor the LDL to induce the artery wall cells to produce monocytechemotactic activity. We also demonstrated that after injection of apoA-I into mice or infusion into humans, the LDL isolated from the mice orhuman volunteers was resistant to oxidation by human artery wall cellsand did not induce monocyte chemotactic activity in the artery wall cellcocultures.

[0062] Without being bound to a particular theory, we believe thepeptides of this invention function in a manner similar to the activityof the apo A-I mimetics described in PCT publication WO 02/15923. Inparticular, we believe the present invention functions in part byincreasing the ant-inflammatory properties of HDL. In particular, webelieve the peptides of this invention bind seeding molecules in LDLthat are necessary for LDL oxidation and then carry the seedingmolecules away where there are ultimately excreted.

[0063] We have discovered that peptides that mimic the amphipathichelical domain(s) of apolipoprotein J are particularly effective inprotecting LDL against oxidation by arterial wall cells and in reducingLDL-induced monocyte chemotactic activity that results from theoxidation of LDL by human artery wall cells. Apo J possesses a widenonpolar face termed globular proteinlike, or G* amphipathic helicaldomains. The class G amphipathic helix is found in globular proteins,and thus, the name class G. The feature of this class of amphipathichelix is that it possesses a random/haphazard distribution of positivelycharged and negatively charged residues on the polar face with a narrownonpolar face. Because of the narrow nonpolar face this class does notreadily associate with phospholipid (see Segrest et al. (1990) Proteins:Structure, Function, and Genetics. 8: 103-117; also see Erratum (1991)Proteins: Structure, Function and Genetics, 9: 79). Several exchangeableapolipoproteins possess similar but not identical characteristics to theG amphipathic helix. Similar to the class G amphipathic helix, thisother class possesses a random distribution of positively and negativelycharged residues on the polar face. However, in contrast to the class Gamphipathic helix which has a narrow nonpolar face, this class has awide nonpolar face that allows this class to readily bind phospholipidand the class is termed G* to differentiate it from the G class ofamphipathic helix (see Segrest et al. (1992) J. Lipid Res., 33: 141-166;also see Anantharamaiah et al. (1993) Pp. 109-142 In The AmphipathicHelix, Epand, R. M. Ed., CRC Press, Boca Raton, Fla.).

[0064] It was a surprising discovery of this invention that theamphipathic helical peptides of this invention required to render humanartery wall cells incapable of oxidizing LDL was substantially less thanthat required for apo AI mimetic peptides such as D4F in a preincubationwith artery wall cells.

[0065] We have demonstrated that oral administration of an apo AImimetic peptide synthesized from D amino acids dramatically reducesatherosclerosis in mice independent of changes in plasma or HDLcholesterol concentrations. Similar to the action of the apo A-Imimetics, we believe that synthetic peptides mimicking the amphipathichelical domains of apo J that are synthesized from D amino acids can begiven orally or by other routes including injection and will ameliorateatherosclerosis and other chronic inflammatory conditions.

[0066] The peptides of this invention can comprise all L-form aminoacids. However, the inventors believe peptides comprising one or moreD-form amino acids and preferably all D-form amino acids (allenantiomeric amino acids are D form) provide for more effective deliveryvia oral administration and will be more stable in the circulation.Particularly preferred peptides are blocked at one or both termini (e.g.with the N-terminus acetylated and the C-terminus amidated).

[0067] The protective function of the peptides of this invention isillustrated in Example 1. The in vitro concentration of the new class ofpeptides necessary to prevent LDL-induced monocyte chemotactic activityby human artery wall cells is 10 to 25 times less than the concentrationrequired for an apoA-I mimetic (D4F) (compare DJ336 to D4F in FIG. 1).Similarly, in a preincubation the peptides of this invention were 1025times more potent in preventing LDL oxidation by artery wall cells(compare DJ336 to D4F in FIG. 2). As shown in FIG. 3, when DJ335 wasgiven orally to LDL receptor null mice it was essentially as effectiveas D4F in rendering HDL more protective in preventing LDL-inducedmonocyte chemotactic activity.

[0068]FIG. 4 demonstrates that when added to the drinking water apeptide of this invention (DJ336) was as potent as D4F in enhancing HDLprotective capacity in apo E null mice. FIG. 5 demonstrates that, whenadded to the drinking water, a peptide of this invention DJ336 wasslightly more potent than D4F in rendering the LDL from apo E null miceresistant to oxidation by human artery wall cells as determined by theinduction of monocyte chemotactic activity. FIG. 6 demonstrates thatwhen added to the drinking water DJ336 was as potent as D4F in causingHDL to inhibit the oxidation of a phospholipid PAPC by the oxidant HPODEin a human artery wall coculture as measured by the generation ofmonocyte chemotactic activity (see Navab et al. (2001) J. Lipid Res. 42:1308-1317 for an explanation of the test system). FIG. 7 demonstratesthat, when added to the drinking water, DJ336 was at least as potent asD4F in increasing the paraoxonase activity of apo E null mice.

[0069] Since many inflammatory conditions have been suspected to bemediated at least in part by oxidized lipids, we believe that thisinvention is also effective in ameliorating conditions that are known orsuspected to be due to the formation of oxidized lipids. These include,but are not limited to, rheumatoid arthritis, lupus erythematous,polyarteritis nodosa, and osteoporosis.

[0070] Without being bound to a particular theory, we believeadministration (e.g. injection) of one or more of the peptides of thisinvention will ameliorate the signs and symptoms of influenza A. Inaddition, the peptide will dramatically reduced the influx ofmacrophages into the artery wall. This will have great utility inreducing the high rate of heart attack and stroke after influenza andother viral infections. Thus, the peptides of this invention can be usedto ameliorate the signs and symptoms of influenza and various otherviral illnesses and reduce the incidence of heart attack and stroke thatoften follows these viral illnesses.

[0071] In view of the foregoing, in one embodiment, this inventionprovides methods for ameliorating and/or preventing one or more symptomsof atherosclerosis and/or a pathology associated with (characterized by)an inflammatory response. The methods typically involve administering toan organism, preferably a mammal, more preferably a human one or more ofthe peptides of this invention (or mimetics of such peptides). Thepeptide(s) can be administered, as described herein, according to any ofa number of standard methods including, but not limited to injection,suppository, nasal spray, time-release implant, transdermal patch, andthe like. In one particularly preferred embodiment, the peptide(s) areadministered orally (e.g. as a syrup, capsule, or tablet).

[0072] The methods involve the administration of a single polypeptide ofthis invention or the administration of two or more differentpolypeptides. The polypeptides can be provided as monomers or indimeric, oligomeric or polymeric forms. In certain embodiments, themultimeric forms may comprise associated monomers (e.g. ionically orhydrophobically linked) while certain other multimeric forms comprisecovalently linked monomers (directly linked or through a linker).

[0073] While the invention is described with respect to use in humans,it is also suitable for animal, e.g. veterinary use. Thus preferredorganisms include, but are not limited to humans, non-human primates,canines, equines, felines, porcines, ungulates, largomorphs, and thelike.

[0074] The methods of this invention are not limited to humans ornon-human animals showing one or more symptom(s) of atherosclerosis(e.g. hypertension, plaque formation and rupture, reduction in clinicalevents such as heart attack, angina, or stroke, high levels of plasmacholesterol, high levels of low density lipoprotein, high levels of verylow density lipoprotein, or inflammatory proteins, etc.), but are usefulin a prophylactic context. Thus, the peptides of this invention (ormimetics thereof) may be administered to organisms to prevent theonset/development of one or more symptoms of atherosclerosis.Particularly preferred subjects in this context are subjects showing oneor more risk factors for atherosclerosis (e.g. family history,hypertension, obesity, high alcohol consumption, smoking, high bloodcholesterol, high blood triglycerides, elevated blood LDL, VLDL, IDL, orlow HDL, diabetes, or a family history of diabetes, high blood lipids,heart attack, angina or stroke, etc.).

[0075] In addition to methods of use of the atherosclerosis-inhibitingpeptides of this invention, this invention also provides the peptidesthemselves, the peptides formulated as pharmaceuticals, particularly fororal delivery, and kits for the treatment and/or prevention of one ormore symptoms of atherosclerosis.

[0076] II. Mitigation of a Symptom of Atherosclerosis Associated with anAcute Inflammatory Response.

[0077] The atherosclerosis-inhibiting peptides of this invention arealso useful in a number of other contexts. In particular, we haveobserved that cardiovascular complications (e.g. atherosclerosis,stroke, etc.) frequently accompany or follow the onset of an acute phaseinflammatory response. Such an acute state inflammatory response isoften associated with a recurrent inflammatory disease (e.g., leprosy,tuberculosis, systemic lupus erythematosus, and rheumatoid arthritis), aviral infection (e.g. influenza), a bacterial infection, a fungalinfection, an organ transplant, a wound or other trauma, an implantedprosthesis, a biofilm, and the like.

[0078] It was a surprising discovery of this invention thatadministration of one or more of the peptides described herein, canreduce or prevent the formation of oxidized phospholipids during orfollowing an acute phase response and thereby mitigate or eliminatecardiovascular complications associated with such a condition.

[0079] Thus, for example, we have demonstrated that a consequence ofinfluenza infection is the diminution in paraoxonase and plateletactivating acetylhydrolase activity in the HDL. Without being bound by aparticular theory, we believe that, as a result of the loss of these HDLenzymatic activities and also as a result of the association ofpro-oxidant proteins with HDL during the acute phase response, HDL is nolonger able to prevent LDL oxidation and was no longer able to preventthe LDL-induced production of monocyte chemotactic activity byendothelial cells.

[0080] We observed that in a subject injected with very low dosages ofapo-AI mimetics (e.g. 20 micrograms for mice) daily after infection withthe influenza A virus paraoxonase levels did not fall and thebiologically active oxidized phospholipids were not generated beyondbackground (see, e.g., WO 02/15923, PCT/US01/26497).

[0081] It was surprising discovery that the class of peptides describedherein can act in manner similar to the apo-I mimetics described in WO02/15923. In view of this discovery, it is believed that the peptides ofthis invention can be administered (e.g. orally or by injection) topatients with known coronary artery disease during influenza infectionor other events that can generate an acute phase inflammatory response(e.g. due to viral infection, bacterial infection, trauma, transplant,various autoimmune conditions, etc.) and thus we can prevent by thisshort term treatment the increased incidence of heart attack and strokeassociated with pathologies that generate such inflammatory states.

[0082] Thus, in certain embodiments, this invention contemplatesadministering one or more of the peptides of this invention to a subjectat risk for, or incurring, an acute inflammatory response and/or at riskfor or incurring a symptom of atherosclerosis.

[0083] For example, a person having or at risk for coronary disease mayprophylactically be administered a polypeptide of this invention duringflu season. A person (or animal) subject to a recurrent inflammatorycondition, e.g. rheumatoid arthritis, various autoimmune diseases, etc.,can be treated with a polypeptide of this invention to mitigate orprevent the development of atherosclerosis or stroke. A person (oranimal) subject to trauma, e.g. acute injury, tissue transplant, etc.can be treated with a polypeptide of this invention to mitigate thedevelopment of atherosclerosis or stroke.

[0084] In certain instances such methods will entail a diagnosis of theoccurrence or risk of an acute inflammatory response. The acuteinflammatory response typically involves alterations in metabolism andgene regulation in the liver. It is a dynamic homeostatic process thatinvolves all of the major systems of the body, in addition to theimmune, cardiovascular and central nervous system. Normally, the acutephase response lasts only a few days; however, in cases of chronic orrecurring inflammation, an aberrant continuation of some aspects of theacute phase response may contribute to the underlying tissue damage thataccompanies the disease, and may also lead to further complications, forexample cardiovascular diseases or protein deposition diseases such asamyloidosis.

[0085] An important aspect of the acute phase response is the radicallyaltered biosynthetic profile of the liver. Under normal circumstances,the liver synthesizes a characteristic range of plasma proteins atsteady state concentrations. Many of these proteins have importantfunctions and higher plasma levels of these acute phase reactants (APRs)or acute phase proteins (APPs) are required during the acute phaseresponse following an inflammatory stimulus. Although most APRs aresynthesized by hepatocytes, some are produced by other cell types,including monocytes, endothelial cells, fibroblasts and adipocytes. MostAPRs are induced between 50% and several-fold over normal levels. Incontrast, the major APRs can increase to 1000-fold over normal levels.This group includes serum amyloid A (SAA) and either C-reactive protein(CRP) in humans or its homologue in mice, serum amyloid P component(SAP). So-called negative APRs are decreased in plasma concentrationduring the acute phase response to allow an increase in the capacity ofthe liver to synthesize the induced APRs.

[0086] In certain embodiments, the acute phase response, or risktherefore is evaluated by measuring one or more APPs. Measuring suchmarkers is well known to those of skill in the art, and commercialcompanies exist that provide such measurement (e.g. AGP measured byCardiotech Services, Louisville, Ky.).

[0087] III. Mitigation of a Symptom or Condition Associated withCoronary Calcification and Osteoporosis.

[0088] We have also identified oxidized lipids as a cause of coronarycalcification and osteoporosis. Moreover, without being bound to aparticularly theory, we believe the same mechanisms are involved in thepathogenesis of calcific aortic stenosis.

[0089] Thus, in certain embodiments, this invention contemplates the useof the peptides described herein to inhibit or prevent a symptom of adisease such as polymyalgia rheumatica, polyarteritis nodosa,scleroderma, idiopathic pulmonary fibrosis, chronic obstructivepulmonary disease, Alzheimers Disease, AIDS, coronary calcification,calcific aortic stenosis, osteoporosis, and the like.

[0090] IV. Preferred Peptides and Their Preparation.

[0091] Preferred Peptides.

[0092] It was a discovery of this invention that peptides that mimickingthe amphipathic helical domains of apo J are capable of mitigating oneor more symptoms of atherosclerosis and/or other pathologiescharacterized by an inflammatory response. Apolipoprotein J possesses awide nonpolar face termed globular protein-like, or G* amphipathichelical domains. The class G amphipathic helix is found in globularproteins, and thus, the name class G. This class of amphipathic helix ischaracterized by a random distribution of positively charged andnegatively charged residues on the polar face with a narrow nonpolarface. Because of the narrow nonpolar face this class does not readilyassociate with phospholipids. The G* of amphipathic helix possessessimilar, but not identical, characteristics to the G amphipathic helix.Similar to the class G amphipathic helix, the G* class peptidespossesses a random distribution of positively and negatively chargedresidues on the polar face. However, in contrast to the class Gamphipathic helix which has a narrow nonpolar face, this class has awide nonpolar face that allows this class to readily bind phospholipidand the class is termed G* to differentiate it from the G class ofamphipathic helix.

[0093] A variety of suitable peptides of this invention that are relatedto G* amphipathic helical domains of apo J are illustrated in Table 1.TABLE 1 Preferred peptides for use in this invention related to g*amphipathic helical domains of apo J. Amino Acid Sequence SEQ ID NOLLEQLNEQFNWVSRLANLTEGE 1 LLEQLNEQFNWVSRLANL 2 NELQEMSNQGSKYVNKEIQNAVNGV3 IQNAVNGVKQIKTLIEKTNEE 4 RKTLLSNLEEAKKKKEDALNETRESETKLKEL 5PGVCNETMMALWEECK 6 PCLKQTCMKFYARVCR 7 ECKPCLKQTCMKFYARVCR 8 LVGRQLEEFL 9MNGDRIDSLLEN 10 QQTHMLDVMQD 11 FSRASSIIDELFQD 12 PFLEMTHEAQQAMDI 13PTEFTREGDDD 14 RMKDQCDKCREILSV 15 PSQAKLRRELDESLQVAERLTRKYNELLKSYQ 16LLEQLNEQFNWVSRLANLTQGE 17 DQYYLRVTTVA 18 PSGVTEVVVKLFDS 19PKFMETVAEKALQEYRKKHRE 20

[0094] The peptides of this invention, however, are not limited to G*variants of apo J. Generally speaking G* domains from essentially anyother protein preferably apo proteins are also suitable. The particularsuitability of such proteins can readily be determined using assays forprotective activity (e.g. protecting LDL from oxidation, and the like),e.g. as illustrated herein in the Examples. Some particularly preferredproteins include G* amphipathic helical domains or variants thereof(e.g. conservative substitutions, and the like) of proteins including,but not limited to apo AI, apo AIV, apo E, apo CII, apo CIII, and thelike.

[0095] Certain preferred peptides for related to G* amphipathic helicaldomains related to apoproteins other than apo J are illustrated in Table2. TABLE 2 Peptides for use in this invention related to G* amphipathichelical domains related to apoproteins other than apo J. SEQ ID AminoAcid Sequence NO WDRVKDLATVYVDVLKDSGRDYVSQF 21 (Related to the 8 to 33region of apo AI) VATVMWDYFSQLSNNAKEAVEHLQK 22 (Related to the 7 to 31region of apo AIV) RWELALGRFWDYLRWVQTLSEQVQEEL 23 (Related to the 25 to51 region of apo E) LSSQVTQELRALMDETMKELKELKAYKSELEEQLT 24 (Related tothe 52 to 83 region of apo E) ARLSKELQAAQARLGADMEDVCGRLV 25 (Related tothe 91 to 116 region of apo E) VRLASHLRKLRKRLLRDADDLQKRLA 26 (Related tothe 135 to 160 region of apo E) PLVEDMQRQWAGLVEKVQA 27 (267 to 285 ofapo E.27) MSTYTGIFTDQVLSVLK 28 (Related to the 60 to 76 region of apoCII) LLSFMQGYMKHATKTAKDALSS 29 (Related to the 8 to 29 region of apoCIII)

[0096] While the various peptides listed in Table 1 and Table 2 areshown with no protecting groups, in certain embodiments (e.g.particularly for oral administration), they bear one or two protectinggroups, more preferably terminal protecting groups. Thus, for example,in certain embodiments, any of the peptides descry bed herein can bear,e.g. an acetyl group protecting the amino terminus and/or an amide groupprotecting the carboxyl terminus. One example of such a “dual protectedpeptide is Ac-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-E-G-E-NH₂ (SEQ IDNO:1 with blocking groups), either or both of these protecting groupscan be eliminated and/or substituted with another protecting group asdescribed herein. In particularly preferred embodiments, the peptidescomprise one or more D-form (dextro rather than levo) amino acids asdescribed herein. In certain embodiments at least two enantiomeric aminoacids, more preferably at least 4 enantiomeric amino acids and mostpreferably at least 8 or 10 enantiomeric amino acids are “D” form aminoacids. In certain embodiments every amino acid (e.g. every enantiomericamino acid) of the peptides described herein is a D-form amino acid.

[0097] It is also noted that the peptides listed in Tables 1 and 2 arenot fully inclusive. Using the teaching provided herein, other suitablepeptides can routinely be produced (e.g. by conservative orsemi-conservative substitutions (e.g. D replaced by E), extensions,deletions, and the like). Thus, for example, one embodiment utilizestruncations of any one or more of peptides identified by SEQ IDNos:1-29.

[0098] Longer peptides are also suitable. Such longer peptides mayentirely form a class G or G* amphipathic helix, or the G amphipathichelix (helices) can form one or more domains of the peptide. Inaddition, this invention contemplates multimeric versions of thepeptides. Thus, for example, the peptides illustrated in Tables 1 or 2can be coupled together (directly or through a linker (e.g. a carbonlinker, or one or more amino acids) with one or more intervening aminoacids). Suitable linkers include, but are not limited to Proline(-Pro-), Gly₄Ser₃ (SEQ ID NO:30), and the like. Thus, one illustrativemultimeric peptide according to this invention is (D-J336)-P-(D-J336)(i.e.Ac-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-E-G-E-P-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-E-G-E-NH₂,SEQ ID NO:31).

[0099] This invention also contemplates the use of “hybrid” peptidescomprising a one or more G or G* amphipathic helical domains and one ormore class A amphipathic helices. Suitable class A amphipathic helicalpeptides are described in PCT publication WO 02/15923. Thus, by way ofillustration, one such “hybrid” peptide is (D-J336)-Pro-(4F) (i.e.Ac-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-E-G-E-P-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂,SEQ ID NO:32), and the like. As indicated above, the peptides of thisinvention preferably comprise one or more D amino acids, more preferablywith every amino acid a D amino acid as described herein and/or havingone or both termini protected. Preferably at least 50% of theenantiomeric amino acids are “D” form, more preferably at least 80% ofthe enantiomeric amino acids are “D” form, and most preferably at least90% or even all of the enantiomeric amino acids are “D” form aminoacids.

[0100] It was a surprising discovery of this invention that, when theamphipathic helical peptides of this invention (e.g. as illustrated inFIGS. 3, 4, 5, 6, and 7) incorporated D amino acids they retained theiractivity even when administered orally. Moreover this oraladministration resulted in relatively efficient uptake and significantserum half-life thereby providing an efficacious method of mitigatingone or more symptoms of atherosclerosis and/or other conditionscharacterized by an inflammatory response.

[0101] Using the teaching provided herein, one of skill can routinelymodify the illustrated amphipathic helical peptides to produce othersuitable apo J variants and/or amphipathic G helical peptides of thisinvention. For example, routine conservative or semi-conservativesubstitutions (e.g. E for D) can be made of the existing amino acids.The effect of various substitutions on lipid affinity of the resultingpeptide can be predicted using the computational method described byPalgunachari et al. (1996) Arteriosclerosis, Thrombosis, & VascularBiology 16: 328-338. The peptides can be lengthened or shortened as longas the class helix structure(s) are preserved. In addition,substitutions can be made to render the resulting peptide more similarto peptide(s) endogenously produced by the subject species.

[0102] New peptides can be designed and/or evaluated using computationalmethods. Computer programs to identify and classify amphipathic helicaldomains are well known to those of skill in the art and many have beendescribed by Jones et al.(1992) J. Lipid Res. 33: 287-296). Suchprograms include, but are not limited to the helical wheel program(WHEEL or WHEEL/SNORKEL), helical net program (HELNET, HELNET/SNORKEL,HELNET/Angle), program for addition of helical wheels (COMBO orCOMBO/SNORKEL), program for addition of helical nets (COMNET,COMNET/SNORKEL, COMBO/SELECT, COMBO/NET), consensus wheel program(CONSENSUS, CONSENSUS/SNORKEL), and the like.

[0103] While, in preferred embodiments, the peptides of this inventionutilize naturally-occurring amino acids or D forms of naturallyoccurring amino acids, substitutions with non-naturally occurring aminoacids (e.g., methionine sulfoxide, methionine methylsulfonium,norleucine, episilon-aminocaproic acid, 4-aminobutanoic acid,tetrahydroisoquinoline-3-carboxylic acid, 8-aminocaprylic acid,4-aminobutyric acid, Lys(N(epsilon)-trifluoroacetyl), α-aminoisobutyricacid, and the like) are also contemplated.

[0104] In addition to the G* amphipathic helical peptides describedherein, peptidomimetics are also contemplated herein. Peptide analogsare commonly used in the pharmaceutical industry as non-peptide drugswith properties analogous to those of the template peptide. These typesof non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (Fauchere (1986) Adv. Drug Res. 15: 29; Veber andFreidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem. 30:1229) and are usually developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect.

[0105] Generally, peptidomimetics are structurally similar to a paradigmpolypeptide (e.g. SEQ ID NO:1 shown in Table 1), but have one or morepeptide linkages optionally replaced by a linkage selected from thegroup consisting of: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂—, —CH₂SO—, etc. by methods known in the artand further described in the following references: Spatola (1983) p. 267in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B.Weinstein, eds., Marcel Dekker, New York,; Spatola (1983) Vega Data 1(3)Peptide Backbone Modifications. (general review); Morley (1980) TrendsPharm Sci pp. 463-468 (general review); Hudson et al. (1979) Int J PeptProt Res 14:177-185 (—CH₂NH—, CH₂CH₂—); Spatola et al. (1986) Life Sci38:1243-1249 (—CH₂—S); Hann, (1982) J Chem Soc Perkin Trans I 1307-314(—CH—CH—, cis and trans); Almquist et al. (1980) J Med Chem.23:1392-1398 (—COCH₂—); Jennings-White et al.(1982) Tetrahedron Lett.23:2533 (—COCH₂—); Szelke et al., European Appln. EP 45665 (1982) CA:97:39405 (1982) (—CH(OH)CH2—); Holladay et al. (1983) Tetrahedron Lett24:4401-4404 (—C(OH)CH₂—); and Hruby (1982) Life Sci., 31:189-199(—CH₂—S—)).

[0106] A particularly preferred non-peptide linkage is —CH₂NH—. Suchpeptide mimetics may have significant advantages over polypeptideembodiments, including, for example: more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), reduced antigenicity, and others.

[0107] In addition, circularly permutations of the peptides describedherein or constrained peptides (including cyclized peptides) comprisinga consensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch (1992) Ann. Rev. Biochem. 61: 387); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

[0108] Peptide Preparation.

[0109] The peptides used in this invention can be chemically synthesizedusing standard chemical peptide synthesis techniques or, particularlywhere the peptide does not comprise “D” amino acid residues, can berecombinantly expressed. In certain embodiments, even peptidescomprising “D” amino acid residues are recombinantly expressed. Wherethe polypeptides are recombinantly expressed, a host organism (e.g.bacteria, plant, fungal cells, etc.) in cultured in an environment whereone or more of the amino acids is provided to the organism exclusivelyin a D form. Recombinantly expressed peptides in such a system thenincorporate those D amino acids.

[0110] In preferred embodiments the peptides are chemically synthesizedby any of a number of fluid or solid phase peptide synthesis techniquesknown to those of skill in the art. Solid phase synthesis in which theC-terminal amino acid of the sequence is attached to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence is a preferred method for the chemical synthesis of thepolypeptides of this invention. Techniques for solid phase synthesis arewell known to those of skill in the art and are described, for example,by Barany and Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methodsin Peptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem.Soc., 85: 2149-2156, and Stewart et al. (1984) Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.

[0111] In a most preferred embodiment, the peptides are synthesized bythe solid phase peptide synthesis procedure using a benzhyderylamineresin (Beckman Bioproducts, 0.59 mmol of NH₂/g of resin) as the solidsupport. The COOH terminal amino acid (e.g., t-butylcarbonyl-Phe) isattached to the solid support through a 4-(oxymethyl)phenacetyl group.This is a more stable linkage than the conventional benzyl esterlinkage, yet the finished peptide can still be cleaved by hydrogenation.Transfer hydrogenation using formic acid as the hydrogen donor is usedfor this purpose. Detailed protocols used for peptide synthesis andanalysis of synthesized peptides are described in a miniprint supplementaccompanying Anantharamaiah et al. (1985) J. Biol. Chem., 260(16):10248-10255.

[0112] It is noted that in the chemical synthesis of peptides,particularly peptides comprising D amino acids, the synthesis usuallyproduces a number of truncated peptides in addition to the desiredfull-length product. The purification process (e.g. HPLC) typicallyresults in the loss of a significant amount of the full-length product.

[0113] It was a discovery of this invention that, in the synthesis of aD peptide (e.g. D-4), in order to prevent loss in purifying the longestform one can dialyze and use the mixture and thereby eliminate the lastHPLC purification. Such a mixture loses about 50% of the potency of thehighly purified product (e.g. per wt of protein product), but themixture contains about 6 times more peptide and thus greater totalactivity.

[0114] D-form Amino Acids.

[0115] D-amino acids are incorporated at one or more positions in thepeptide simply by using a D-form derivatized amino acid residue in thechemical synthesis. D-form residues for solid phase peptide synthesisare commercially available from a number of suppliers (see, e.g.,Advanced Chem Tech, Louisville; Nova Biochem, San Diego; Sigma, StLouis; Bachem California Inc., Torrance, etc.). The D-form amino acidscan be incorporated at any position in the peptide as desired. Thus, forexample, in one embodiment, the peptide can comprise a single D-aminoacid, while in other embodiments, the peptide comprises at least two,generally at least three, more generally at least four, most generallyat least five, preferably at least six, more preferably at least sevenand most preferably at least eight D amino acids. In particularlypreferred embodiments, essentially every other (enantiomeric) amino acidis a D-form amino acid. In certain embodiments at least 90%, preferablyat least 90%, more preferably at least 95% of the enantiomeric aminoacids are D-form amino acids. In one particularly preferred embodiment,essentially every enantiomeric amino acid is a D-form amino acid.

[0116] Protecting Groups.

[0117] In certain embodiments, the one or more R-groups on theconstituent amino acids and/or the terminal amino acids are blocked witha protecting group. Without being bound by a particular theory, it was adiscovery of this invention that blockage, particularly of the aminoand/or carboxyl termini of the subject peptides of this inventiongreatly improves oral delivery and significantly increases serumhalf-life.

[0118] A wide number of protecting groups are suitable for this purpose.Such groups include, but are not limited to acetyl, amide, and alkylgroups with acetyl and alkyl groups being particularly preferred forN-terminal protection and amide groups being preferred for carboxylterminal protection. In certain particularly preferred embodiments, theprotecting groups include, but are not limited to alkyl chains as infatty acids, propeonyl, formyl, and others. Particularly preferredcarboxyl protecting groups include amides, esters, and ether-formingprotecting groups. In one preferred embodiment, an acetyl group is usedto protect the amino terminus and an amide group is used to protect thecarboxyl terminus. These blocking groups enhance the helix-formingtendencies of the peptides. Certain particularly preferred blockinggroups include alkyl groups of various lengths, e.g. groups having theformula: CH₃—(CH₂)_(n)—CO— where n ranges from about 1 to about 20,preferably from about 1 to about 16 or 18, more preferably from about 3to about 13, and most preferably from about 3 to about 10.

[0119] In certain particularly preferred embodiments, the protectinggroups include, but are not limited to alkyl chains as in fatty acids,propeonyl, formyl, and others. Particularly preferred carboxylprotecting groups include amides, esters, and ether-forming protectinggroups. In one preferred embodiment, an acetyl group is used to protectthe amino terminus and an amide group is used to protect the carboxylterminus. These blocking groups enhance the helix-forming tendencies ofthe peptides. Certain particularly preferred blocking groups includealkyl groups of various lengths, e.g. groups having the formula:CH₃—(CH₂)_(n)—CO— where n ranges from about 3 to about 20, preferablyfrom about 3 to about 16, more preferably from about 3 to about 13, andmost preferably from about 3 to about 10.

[0120] Other protecting groups include, but are not limited to Fmoc,t-butoxycarbonyl (t-BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylicgroup, 9-florenecarboxylic group, 9-fluorenone-1-carboxylic group,benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt),4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh),Tosyl (Tos),2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl),4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), cyclohexyloxy(cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl(Ac), and Trifluoroacetyl (TFA).

[0121] Protecting/blocking groups are well known to those of skill asare methods of coupling such groups to the appropriate residue(s)comprising the peptides of this invention (see, e.g., Greene et al.,(1991) Protective Groups in Organic Synthesis, 2nd ed., John Wiley &Sons, Inc. Somerset, N.J.). In one preferred embodiment, for example,acetylation is accomplished during the synthesis when the peptide is onthe resin using acetic anhydride. Amide protection can be achieved bythe selection of a proper resin for the synthesis. During the synthesisof the peptides described herein in the examples, rink amide resin wasused. After the completion of the synthesis, the semi-permanentprotecting groups on acidic bifunctional amino acids such as Asp and Gluand basic amino acid Lys, hydroxyl of Tyr are all simultaneouslyremoved. The peptides released from such a resin using acidic treatmentcomes out with the n-terminal protected as acetyl and the carboxylprotected as NH₂ and with the simultaneous removal of all of the otherprotecting groups.

[0122] V. Enhancing Peptide Uptake.

[0123] It was also a surprising discovery of this invention that when anall L amino acid peptide (e.g. otherwise having the sequence of thepeptides of this invention) is administered in conjunction with theD-form (i.e. a peptide of this invention) the uptake of the D-formpeptide is increased. Thus, in certain embodiments, this inventioncontemplates the use of combinations of D-form and L-form peptides inthe methods of this invention. The D-form peptide and the L-form peptidecan have different amino acid sequences, however, in preferredembodiments, they both have amino acid sequences of peptides describedherein, and in still more preferred embodiments, they have the sameamino acid sequence.

[0124] It was also a discovery of this invention that concatamers of theamphipathic helix peptides of this invention are also effective inmitigating one or more symptoms of atherosclerosis. The monomerscomprising the concatamers can be coupled directly together or joined bya linker. In certain embodiments, the linker is an amino acid linker(e.g. a proline), or a peptide linker (e.g. Gly₄Ser₃, SEQ ID NO:30). Incertain embodiments, the concatamer is a 2 mer, more preferably a 3 mer,still more preferably a 4 mer, and most preferably 5 mer, 8 mer or 10mer. As indicated above, the concatamer can comprise a G* relatedamphipathic helix as described herein combined with an apo A-I variantas described in PCT publication WO 02/15923.

[0125] VI. Pharmaceutical Formulations.

[0126] In order to carry out the methods of the invention, one or morepeptides or peptide mimetics of this invention are administered, e.g. toan individual diagnosed as having one or more symptoms ofatherosclerosis, or as being at risk for atherosclerosis. The peptidesor peptide mimetics can be administered in the “native” form or, ifdesired, in the form of salts, esters, amides, prodrugs, derivatives,and the like, provided the salt, ester, amide, prodrug or derivative issuitable pharmacologically, i.e., effective in the present method.Salts, esters, amides, prodrugs and other derivatives of the activeagents may be prepared using standard procedures known to those skilledin the art of synthetic organic chemistry and described, for example, byMarch (1992) Advanced Organic Chemistry; Reactions, Mechanisms andStructure, 4th Ed. N.Y. Wiley-Interscience.

[0127] For example, acid addition salts are prepared from the free baseusing conventional methodology, that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or may be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include both organic acids, e.g., aceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malicacid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. An acid addition salt may be reconvertedto the free base by treatment with a suitable base. Particularlypreferred acid addition salts of the active agents herein are halidesalts, such as may be prepared using hydrochloric or hydrobromic acids.Conversely, preparation of basic salts of the peptides or mimetics areprepared in a similar manner using a pharmaceutically acceptable basesuch as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine, or the like. Particularly preferredbasic salts include alkali metal salts, e.g., the sodium salt, andcopper salts.

[0128] Preparation of esters typically involves functionalization ofhydroxyl and/or carboxyl groups which may be present within themolecular structure of the drug. The esters are typicallyacyl-substituted derivatives of free alcohol groups, i.e., moieties thatare derived from carboxylic acids of the formula RCOOH where R is alky,and preferably is lower alkyl. Esters can be reconverted to the freeacids, if desired, by using conventional hydrogenolysis or hydrolysisprocedures.

[0129] Amides and prodrugs may also be prepared using techniques knownto those skilled in the art or described in the pertinent literature.For example, amides may be prepared from esters, using suitable aminereactants, or they may be prepared from an anhydride or an acid chlorideby reaction with ammonia or a lower alkyl amine. Prodrugs are typicallyprepared by covalent attachment of a moiety that results in a compoundthat is therapeutically inactive until modified by an individual'smetabolic system.

[0130] The peptides or mimetics identified herein are useful forparenteral, topical, oral, nasal (or otherwise inhaled), rectal, orlocal administration, such as by aerosol or transdermally, forprophylactic and/or therapeutic treatment of atherosclerosis and/orsymptoms thereof. The pharmaceutical compositions can be administered ina variety of unit dosage forms depending upon the method ofadministration. Suitable unit dosage forms, include, but are not limitedto powders, tablets, pills, capsules, lozenges, suppositories, patches,nasal sprays, injectibles, implantable sustained-release formulations,lipid complexes, etc.

[0131] The peptides and/or peptide mimetics of this invention aretypically combined with a pharmaceutically acceptable carrier(excipient) to form a pharmacological composition. Pharmaceuticallyacceptable carriers can contain one or more physiologically acceptablecompound(s) that act, for example, to stabilize the composition or toincrease or decrease the absorption of the active agent(s).Physiologically acceptable compounds can include, for example,carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins, protection and uptake enhancers such as lipids, compositionsthat reduce the clearance or hydrolysis of the active agents, orexcipients or other stabilizers and/or buffers.

[0132] Other physiologically acceptable compounds include wettingagents, emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysio-chemical characteristics of the active agent(s).

[0133] The excipients are preferably sterile and generally free ofundesirable matter. These compositions may be sterilized byconventional, well-known sterilization techniques.

[0134] In therapeutic applications, the compositions of this inventionare administered to a patient suffering from one or more symptoms ofatherosclerosis or at risk for atherosclerosis in an amount sufficientto cure or at least partially prevent or arrest the disease and/or itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. In any event, the composition shouldprovide a sufficient quantity of the active agents of the formulationsof this invention to effectively treat (ameliorate one or more symptoms)the patient.

[0135] The concentration of peptide or mimetic can vary widely, and willbe selected primarily based on fluid volumes, viscosities, body weightand the like in accordance with the particular mode of administrationselected and the patient's needs. Concentrations, however, willtypically be selected to provide dosages ranging from about 0.1 or 1mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosagesrange from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably fromabout 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about7.2 mg/kg/day to about 11.0 mg/kg/day, and most preferably from about11.0 mg/kg/day to about 15.0 mg/kg/day. In certain preferredembodiments, dosages range from about 10 mg/kg/day to about 50mg/kg/day. In certain embodiments, dosages range from about 20 mg toabout 50 mg given orally twice daily. It will be appreciated that suchdosages may be varied to optimize a therapeutic regimen in a particularsubject or group of subjects.

[0136] In certain preferred embodiments, the peptides or peptidemimetics of this invention are administered orally (e.g. via a tablet)or as an injectable in accordance with standard methods well known tothose of skill in the art. In other preferred embodiments, the peptides,may also be delivered through the skin using conventional transdermaldrug delivery systems, i.e., transdermal “patches” wherein the activeagent(s) are typically contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the drug composition is typically contained in a layer, or“reservoir,” underlying an upper backing layer. It will be appreciatedthat the term “reservoir” in this context refers to a quantity of“active ingredient(s)” that is ultimately available for delivery to thesurface of the skin. Thus, for example, the “reservoir” may include theactive ingredient(s) in an adhesive on a backing layer of the patch, orin any of a variety of different matrix formulations known to those ofskill in the art. The patch may contain a single reservoir, or it maycontain multiple reservoirs.

[0137] In one embodiment, the reservoir comprises a polymeric matrix ofa pharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

[0138] Other preferred formulations for topical drug delivery include,but are not limited to, ointments and creams. Ointments are semisolidpreparations which are typically based on petrolatum or other petroleumderivatives. Creams containing the selected active agent, are typicallyviscous liquid or semisolid emulsions, often either oil-in-water orwater-in-oil. Cream bases are typically water-washable, and contain anoil phase, an emulsifier and an aqueous phase. The oil phase, alsosometimes called the “internal” phase, is generally comprised ofpetrolatum and a fatty alcohol such as cetyl or stearyl alcohol; theaqueous phase usually, although not necessarily, exceeds the oil phasein volume, and generally contains a humectant. The emulsifier in a creamformulation is generally a nonionic, anionic, cationic or amphotericsurfactant. The specific ointment or cream base to be used, as will beappreciated by those skilled in the art, is one that will provide foroptimum drug delivery. As with other carriers or vehicles, an ointmentbase should be inert, stable, nonirritating and nonsensitizing.

[0139] Unlike typical peptide formulations, the peptides of thisinvention comprising D-form amino acids can be administered, evenorally, without protection against proteolysis by stomach acid, etc.Nevertheless, in certain embodiments, peptide delivery can be enhancedby the use of protective excipients. This is typically accomplishedeither by complexing the polypeptide with a composition to render itresistant to acidic and enzymatic hydrolysis or by packaging thepolypeptide in an appropriately resistant carrier such as a liposome.Means of protecting polypeptides for oral delivery are well known in theart (see, e.g., U.S. Pat. No. 5,391,377 describing lipid compositionsfor oral delivery of therapeutic agents).

[0140] Elevated serum half-life can be maintained by the use ofsustained-release protein “packaging” systems. Such sustained releasesystems are well known to those of skill in the art. In one preferredembodiment, the ProLease biodegradable microsphere delivery system forproteins and peptides (Tracy (1998) Biotechnol. Prog. 14: 108; Johnsonet al. (1996), Nature Med. 2: 795; Herbert et al. (1998), Pharmaceut.Res. 15, 357) a dry powder composed of biodegradable polymericmicrospheres containing the protein in a polymer matrix that can becompounded as a dry formulation with or without other agents.

[0141] The ProLease microsphere fabrication process was specificallydesigned to achieve a high protein encapsulation efficiency whilemaintaining protein integrity. The process consists of (i) preparationof freeze-dried protein particles from bulk protein by sprayfreeze-drying the drug solution with stabilizing excipients, (ii)preparation of a drug-polymer suspension followed by sonication orhomogenization to reduce the drug particle size, (iii) production offrozen drug-polymer microspheres by atomization into liquid nitrogen,(iv) extraction of the polymer solvent with ethanol, and (v) filtrationand vacuum drying to produce the final dry-powder product. The resultingpowder contains the solid form of the protein, which is homogeneouslyand rigidly dispersed within porous polymer particles. The polymer mostcommonly used in the process, poly(lactide-co-glycolide) (PLG), is bothbiocompatible and biodegradable.

[0142] Encapsulation can be achieved at low temperatures (e.g., −40°C.). During encapsulation, the protein is maintained in the solid statein the absence of water, thus minimizing water-induced conformationalmobility of the protein, preventing protein degradation reactions thatinclude water as a reactant, and avoiding organic-aqueous interfaceswhere proteins may undergo denaturation. A preferred process usessolvents in which most proteins are insoluble, thus yielding highencapsulation efficiencies (e.g., greater than 95%).

[0143] In another embodiment, one or more components of the solution canbe provided as a “concentrate”, e.g., in a storage container (e.g., in apremeasured volume) ready for dilution, or in a soluble capsule readyfor addition to a volume of water.

[0144] The foregoing formulations and administration methods areintended to be illustrative and not limiting. It will be appreciatedthat, using the teaching provided herein, other suitable formulationsand modes of administration can be readily devised.

[0145] VII. Lipid-Based Formulations.

[0146] In certain embodiments, the peptides of this invention areadministered in conjunction with one or more lipids. The lipids can beformulated as an excipient to protect and/or enhance transport/uptake ofthe peptides or they can be administered separately.

[0147] Without being bound by a particular theory, it was discovered ofthis invention that administration (e.g. oral administration) of certainphospholipids can significantly increase HDL/LDL ratios. In addition, itis believed that certain medium-length phospholipids are transported bya process different than that involved in general lipid transport. Thus,co-administration of certain medium-length phospholipids with thepeptides of this invention confer a number of advantages: They protectthe phospholipids from digestion or hydrolysis, they improve peptideuptake, and they improve HDL/LDL ratios.

[0148] The lipids can be formed into liposomes that encapsulate thepolypeptides of this invention and/or they can be complexed/admixed withthe polypeptides and/or they can be covalently coupled to thepolypeptides. Methods of making liposomes and encapsulating reagents arewell known to those of skill in the art (see, e.g., Martin andPapahadjopoulos (1982) J. Biol. Chem., 257: 286-288; Papahadjopoulos etal. (1991) Proc. Natl. Acad. Sci. USA, 88: 11460-11464; Huang et al.(1992) Cancer Res., 52:6774-6781; Lasic et al. (1992) FEBS Lett., 312:255-258., and the like).

[0149] Preferred phospholipids for use in these methods have fatty acidsranging from about 4 carbons to about 24 carbons in the sn-I and sn-2positions. In certain preferred embodiments, the fatty acids aresaturated. In other preferred embodiments, the fatty acids can beunsaturated. Various preferred fatty acids are illustrated in Table 3.TABLE 3 Preferred fatty acids in the sn-1 and/or sn-2 position of thepreferred phospholipids for admission of D polypeptides. Carbon No.Common Name IUPAC Name  3:0 Propionoyl Trianoic  4:0 Butanoyl Tetranoic 5:0 Pentanoyl Pentanoic  6:0 Caproyl Hexanoic  7:0 Heptanoyl Heptanoic 8:0 Capryloyl Octanoic  9:0 Nonanoyl Nonanoic 10:0 Capryl Decanoic 11:0Undcanoyl Undecanoic 12:0 Lauroyl Dodecanoic 13:0 TridecanoylTridecanoic 14:0 Myristoyl Tetradecanoic 15:0 PentadecanoylPentadecanoic 16:0 Palmitoyl Hexadecanoic 17:0 HeptadecanoylHeptadecanoic 18:0 Stearoyl Octadecanoic 19:0 Nonadecanoyl Nonadecanoic20:0 Arachidoyl Eicosanoic 21:0 Heniecosanoyl Heniecosanoic 22:0Behenoyl Docosanoic 23:0 Truisanoyl Trocosanoic 24:0 LignoceroylTetracosanoic 14:1 Myristoleoyl (9-cis) 14:1 Myristelaidoyl (9- trans)16:1 Palmitoleoyl (9-cis) 16:1 Palmitelaidoyl (9- trans)

[0150] The fatty acids in these positions can be the same or different.Particularly preferred phospholipids have phosphorylcholine at the sn-3position.

[0151] VIII. Additional Pharmacologically Active Agents.

[0152] Additional pharmacologically active agents may be delivered alongwith the primary active agents, e.g., the peptides of this invention. Inone embodiment, such agents include, but are not limited to agents thatreduce the risk of atherosclerotic events and/or complications thereof.Such agents include, but are not limited to beta blockers, beta blockersand thiazide diuretic combinations, statins, aspirin, ace inhibitors,ace receptor inhibitors (ARBs), and the like.

[0153] Suitable beta blockers include, but are not limited tocardioselective (selective beta 1 blockers), e.g., acebutolol(Sectral™), atenolol (Tenormin™), betaxolol (Kerlone™), bisoprolol(Zebeta™), metoprolol (Lopressor™), and the like. Suitable non-selectiveblockers (block beta 1 and beta 2 equally) include, but are not limitedto carteolol (Cartrol™), nadolol (Corgard™), penbutolol (Levatol™),pindolol (Visken™), propranolol (Inderal™), timolol (Blockadren™),labetalol (Normodyne™, Trandate™), and the like.

[0154] Suitable beta blocker thiazide diuretic combinations include, butare not limited to Lopressor HCT, ZIAC, Tenoretic, Corzide, Timolide,Inderal LA 40/25, Inderide, Normozide, and the like.

[0155] Suitable statins include, but are not limited to pravastatin(Pravachol/Bristol-Myers Squibb), simvastatin (Zocor/Merck), lovastatin(Mevacor/Merck), and the like.

[0156] Suitable ace inhibitors include, but are not limited to captopril(e.g. Capoten™ by Squibb), benazepril (e.g., Lotensin™ by Novartis),enalapril (e.g., Vasotec™ by Merck), fosinopril (e.g., Monopril™ byBristol-Myers), lisinopril (e.g. Prinivil™ by Merck or Zestril™ byAstra-Zeneca), quinapril (e.g. Accupril™ by Parke-Davis), ramipril(e.g., Altace™ by Hoechst Marion Roussel, King Pharmaceuticals),imidapril, perindopril erbumine (e.g., Aceon™ by Rhone-Polenc Rorer),trandolapril (e.g., Mavik™ by Knoll Pharmaceutical), and the like.Suitable ARBS (Ace Receptor Blockers) include but are not limited tolosartan (e.g. Cozaar™ by Merck), irbesartan (e.g., Avapro™ by Sanofi),candesartan (e.g., Atacand™ by Astra Merck), valsartan (e.g., Diovan™ byNovartis), and the like.

[0157] IX. Kits for the Amelioration of One or More Symptoms ofAtherosclerosis.

[0158] In another embodiment this invention provides kits foramelioration of one or more symptoms of atherosclerosis or for theprophylactic treatment of a subject (human or animal) at risk foratherosclerosis. The kits preferably comprise a container containing oneor more of the peptides or peptide mimetics of this invention. Thepeptide or peptide mimetic can be provided in a unit dosage formulation(e.g. suppository, tablet, caplet, patch, etc.) and/or may be optionallycombined with one or more pharmaceutically acceptable excipients.

[0159] The kit can, optionally, further comprise one or more otheragents used in the treatment of heart disease and/or atherosclerosis.Such agents include, but are not limited to, beta blockers,vasodilators, aspirin, statins, ace inhibitors or ace receptorinhibitors (ARBs) and the like, e.g. as described above.

[0160] In addition, the kits optionally include labeling and/orinstructional materials providing directions (i.e., protocols) for thepractice of the methods or use of the “therapeutics” or “prophylactics”of this invention. Preferred instructional materials describe the use ofone or more polypeptides of this invention to mitigate one or moresymptoms of atherosclerosis and/or to prevent the onset or increase ofone or more of such symptoms in an individual at risk foratherosclerosis and/or to mitigate one or more symptoms of a pathologycharacterized by an inflammatory response. The instructional materialsmay also, optionally, teach preferred dosages/therapeutic regiment,counter indications and the like.

[0161] While the instructional materials typically comprise written orprinted materials they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

EXAMPLES

[0162] The following examples are offered to illustrate, but not tolimit the claimed invention.

Example 1 Use of ApoJ-Related Peptides to Mediate Symptoms ofAtherosclerosis

[0163] Prevention of LDL-Induced Monocyte Chemotactic Activity

[0164]FIG. 1 illustrates a comparison of the effect of D-4F (Circulation2002; 105:290-292) with the effect of an apoJ peptide made from D aminoacids (D-J336, Ac-LLEQLNEQFNWVSRLANLTEGE-NH₂, SEQ ID NO:1) on theprevention of LDL-induced monocyte chemotactic activity in vitro in aco-incubation. Human aortic endothelial cells were incubated with mediumalone (no addition), with control human LDL (200 μg protein/ml) orcontrol human LDL+control human HDL (350 μg HDL protein/ml). D-J336 orD-4F was added to other wells in a concentration range as indicated pluscontrol human LDL (200 μg protein/ml). Following overnight incubation,the supernatants were assayed for monocyte chemotactic activity. Asshown in FIG. 1, the in vitro concentration of the apoJ variant peptidethat prevents LDL-induced monocyte chemotactic activity by human arterywall cells is 10 to 25 times less than the concentration required forthe D-4F peptide.

[0165] Prevention of LDL-Induced Monocyte Chemotactic Activity byPre-Treatment of Artery Wall Cells with D-J336

[0166]FIG. 2 illustrates a comparison of the effect of D-4F with theeffect of D-J336 on the prevention of LDL induced monocyte chemotacticactivity in a pre-incubation. Human aortic endothelial cells werepre-incubated with D-J336 or D-4F at 4, 2, and 1 μg/ml for DJ336 or 100,50, 25, and 12.5 μg/ml for D-4F for 6 hrs. The cultures were then washedand were incubated with medium alone (no addition), or with controlhuman LDL (200 μg protein/ml), or with control human LDL+control humanHDL (350 μg HDL protein/ml) as assay controls. The wells that werepre-treated with peptides received the control human LDL at 200 μgprotein/ml. Following overnight incubation, the supernatants wereassayed for monocyte chemotactic activity.

[0167] As illustrated in FIG. 2, the ApoJ variant peptide was 10-25times more potent in preventing LDL oxidation by artery wall cells invitro. The effect of apo J peptide mimetics on HDL protective capacityin LDL receptor null mice.

[0168] D-4F designated as F, or the apoJ peptide made from D amino acids(D-J336, designated as J) was added to the drinking water of LDLreceptor null mice (4 per group) at 0.25 or 0.5 mg per ml of drinkingwater. After 24- or 48-hrs blood was collected from the mice and theirHDL was isolated and tested for its ability to protect againstLDL-induced monocyte chemotactic activity. Assay controls includedculture wells that received no lipoproteins (no addition), or controlhuman LDL alone (designated as LDL, 200 μg cholesterol/ml), or controlLDL+control human HDL (designated as +HDL, 350 μg HDL cholesterol). Fortesting the mouse HDL, the control LDL was added together with mouse HDL(+F HDL or +J HDL) to artery wall cell cultures. The mouse HDL was addedat 100 μg cholesterol/ml respectively. After treatment with either D-4For D-J336 the mouse HDL at 100 μg/ml was as active as 350 μg/ml ofcontrol human HDL in preventing the control LDL from inducing the arterywall cells to produce monocyte chemotactic activity. The reason for thediscrepancy between the relative doses required for the D-J336 peptiderelative to D-4F in vitro and in vivo may be related to the solubilityof the peptides in water and we believe that when measures are taken toachieve equal solubility the D-J peptides will be much more active invivo as they are in vitro.

[0169] Protection Against LDL-Induced Monocyte Chemotactic Activity byHDL from apo E Null Mice given Oral Peptides.

[0170]FIG. 4 illustrates the effect of oral apoA-1 peptide mimetic andapoj peptide on HDL protective capacity. ApoE null mice (4 per group)were provided with D-4F (designated as F) at 50, 30, 20, 10, 5 μg per mlof drinking water or apoJ peptide (designated as J) at 50, 30 or 20 μgper ml of drinking water. After 24 hrs blood was collected, plasmafractionated by FPLC and fractions containing LDL (designated as mlDLfor murine LDL) and fractions containing HDL (designated as mHDL) wereseparately pooled and HDL protective capacity against LDL oxidation asdetermined by LDL-induced monocyte chemotactic activity was determined.For the assay controls the culture wells received no lipoproteins (noadditions), mlDL alone (at 200 μg cholesterol/ml), or mlDL+standardnormal human HDL (designated as Cont. h HDL, at 350 μg HDLcholesterol/ml).

[0171] For testing the murine HDL, mlDL together with murine HDL (+FmHDL or +J mHDL) were added to artery wall cell cultures. The HDL fromthe mice that did not receive any peptide in their drinking water isdesignated as no peptide mHDL. The murine HDL was used at 100 μgcholesterol/ml. After receiving D-4F or D-J336 the murine HDL at 100μg/ml was as active as 350 μg/ml of normal human HDL. As shown in FIG.4, when added to the drinking water the D-J peptide was as potent asD-4F in enhancing HDL protective capacity in apo E null mice.

[0172] Ability of LDL Obtained from apoE Null Mice Given Oral Peptidesto Induce Monocyte Chemotactic Activity.

[0173]FIG. 5 illustrates the effect of oral apo A-1 peptide mimetic andapoJ peptide on LDL susceptibility to oxidation. ApoE null mice (4 pergroup) were provided, in their drinking water, with D-4F (designated asF) at 50, 30, 20, 10, 5 μg per ml of drinking water or the apoJ peptide(D-J336 made from D amino acids and designated as J) at 50, 30 or 20 μgper ml of drinking water. After 24 hrs blood was collected from the miceshown in FIG. 4, plasma fractionated by FPLC and fractions containingLDL (designated as mlDL for murine LDL) were pooled and LDLsusceptibility to oxidation as determined by induction of monocytechemotactic activity was determined. For the assay controls the culturewells received no lipoproteins (no additions), mlDL alone (at 200 μgcholesterol/ml), or mlDL+standard normal human HDL (designated as Cont.h HDL, 350 μg HDL cholesterol).

[0174] Murine LDL, mlDL, from mice that received the D-4F (F mlDL) orthose that received the apoJ peptide (J mlDL) were added to artery wallcell cultures. LDL from mice that did not receive any peptide in theirdrinking water is designated as No peptide LDL.

[0175] As shown in FIG. 5, when added to the drinking water, D-J336 wasslightly more potent than D-4F in rendering the LDL from apo E null miceresistant to oxidation by human artery wall cells as determined by theinduction of monocyte chemotactic activity.

[0176] Protection Against Phospholipid Oxidation and Induction ofMonocyte Chemotactic Activity by HDL Obtained from apo E Null Mice GivenOral Peptides.

[0177]FIG. 6 illustrates the effect of oral apoA-1 peptide mimetic andapoj peptide on HDL protective capacity. ApoE null mice (4 per group)were provided with D-4F (designated as F) at 50, 30, 20, 10, 5 μg per mlof drinking water or apoj peptide (D-J336 made from D amino acids anddesignated as J) at 50, 30 or 20 μg per ml of drinking water. After 24hrs blood was collected, plasma fractionated by FPLC and fractionscontaining HDL (designated as mHDL) were pooled and HDL protectivecapacity against PAPC oxidation as determined by the induction ofmonocyte chemotactic activity was determined. For the assay controls theculture wells received no lipoproteins (no additions), the phospholipidPAPC at 20 μg/ml+HPODE, at 1.0 μg/ml, or PAPC+HPODE plus standard normalhuman HDL (at 350 μg HDL cholesterol/ml and designated as+Cont. h HDL).

[0178] For testing the murine HDL, PAPC+HPODE together with murine HDL(+F mHDL or +J mHDL) were added to artery wall cell cultures. The HDLfrom mice that did not receive any peptide in their drinking water isdesignated as “no peptide mHDL”. The murine HDL was used at 100 μgcholesterol/ml.

[0179] The data show in FIG. 6 indicate that, when added to the drinkingwater, D-J336 was as potent as D-4F in causing HDL to inhibit theoxidation of a phospholipid PAPC by the oxidant HPODE in a human arterywall co-culture as measured by the generation of monocyte chemotacticactivity

[0180] Effect of Oral apoA-1 Peptide Mimetic and apoJ Peptide on PlasmaParaoxonase Activity in Mice.

[0181]FIG. 7 shows the effect of oral apoA-1 peptide mimetic and apojpeptide on plasma paraoxonase activity in mice. ApoE null mice (4 pergroup) were provided with D-4F designated as F at 50, 10, 5 or 0 μg perml of drinking water or apoJ peptide (D-J336 made from D amino acids anddesignated as J) at 50, 10 or 5 μg per ml of drinking water. After 24hrs blood was collected and plasma was assayed for PON1 activity. Thesedata demonstrate that, when added to the drinking water, D-J336 was atleast as potent as D-4F in increasing the paraoxonase activity of apo Enull mice.

[0182] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

What is claimed is:
 1. An isolated polypeptide that ameliorates asymptom of atherosclerosis or other pathology associated with aninflammatory response, said polypeptide comprising an amphipathichelical peptide having charged residues on the polar face of the peptideand possessing a wide non-polar face.
 2. The polypeptide of claim 1,wherein said peptide is at least 10 amino acids in length.
 3. Thepolypeptide of claim 2, wherein said peptide is about 40 or fewerpeptides in length.
 4. The polypeptide of claim 2, wherein said peptidecomprises a G* amphipathic helix.
 5. The polypeptide of claim 4, whereinsaid peptide shows greater than about 50% sequence identity with apo J.6. The polypeptide of claim 2, wherein said peptide protects aphospholipid against oxidation by an oxidizing agent.
 7. The polypeptideof claim 2, wherein said peptide comprises an amino acid sequenceselected from the group consisting of LLEQLNEQFNWVSRLANLTEGE, (SEQ IDNO:1), LLEQLNEQFNWVSRLANL, (SEQ ID NO:2), NELQEMSNQGSKYVNKEIQNAVNGV,(SEQ ID NO:3), IQNAVNGVKQIKTLIEKTNEE, (SEQ ID NO:4),RKTLLSNLEEAKKKKEDALNETRESETKLKEL, (SEQ ID NO:5), PGVCNETMMALWEECK, (SEQID NO:6), PCLKQTCMKFYARVCR, (SEQ ID NO:7), ECKPCLKQTCMKFYARVCR, (SEQ IDNO:8), LVGRQLEEFL, (SEQ ID NO:9), MNGDRIDSLLEN, (SEQ ID NO:10),QQTHMLDVMQD, (SEQ ID NO:11), FSRASSIIDELFQD, (SEQ ID NO:12),PFLEMIHEAQQAMDI, (SEQ ID NO:13), PTEFIREGDDD, (SEQ ID NO:14),RMKDQCDKCREILSV, (SEQ ID NO:15), PSQAKLRRELDESLQVAERLTRKYNELLKSYQ, (SEQID NO:16), LLEQLNEQFNWVSRLANLTQGE, (SEQ ID NO:17), DQYYLRVTTVA, (SEQ IDNO:18), PS GVTEVVVKLFDS, (SEQ ID NO:19), PKFMETVAEKALQEYRKKHRE, (SEQ IDNO:20), WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO:21),VATVMWDYFSQLSNNAKEAVEHLQK (SEQ ID NO:22), RWELALGRFWDYLRWVQTLSEQVQEEL(SEQ ID NO:23), LSSQVTQELRALMDETMKELKELKAYKSELEEQLT (SEQ ID NO:24),ARLSKELQAAQARLGADMEDVCGRLV (SEQ ID NO:25), VRLASHLRKLRKRLLRDADDLQKRLA(SEQ ID NO:26), PLVEDMQRQWAGLVEKVQA (SEQ ID NO:27), MSTYTGIFTDQVLSVLK(SEQ ID NO:28), and LLSFMQGYMKHATKTAKDALSS (SEQ ID NO:29).
 8. Thepolypeptide of claim 7, wherein said peptide is a concatamer of two ormore of said amino acid sequences.
 9. The polypeptide of claim 2,wherein said peptide further comprises a protecting group.
 10. Thepolypeptide of claim 2, wherein said peptide further comprises aprotecting group coupled to the amino or carboxyl terminus.
 11. Thepolypeptide of claim 9, wherein said protecting group is a protectinggroup selected from the group consisting of acetyl, amide, 3 to 20carbon alkyl groups, Fmoc, t-boc, 9-fluoreneacetyl group,1-fluorenecarboxylic group, 9-florenecarboxylic group,9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan),Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt),4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl(Mts), 4,4=-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl(MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom),t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum),t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), a benzoyl group, acarbobenzoxy group, a propyl group, a butyl group, a pentyl group, ahexyl group group, and Trifluoroacetyl (TFA).
 12. The polypeptide ofclaim 9, wherein said peptide comprises a protecting group coupled tothe amino terminal and said amino terminal protecting group is aprotecting group selected from the group consisting of a benzoyl group,an acetyl, a propeonyl, a carbobenzoxy, a propyl, a butyl, a pentyl, ahexyl, and a 3 to 20 carbon alkyl.
 13. The polypeptide of claim 9,wherein said peptide comprises a protecting group coupled to thecarboxyl terminal and said carboxyl terminal protecting group is anamide.
 14. The polypeptide of claim 9, wherein said peptide furthercomprises: a first protecting group coupled to the amino terminuswherein said protecting group is a protecting group selected from thegroup consisting of a benzoyl group, an acetyl, a propeonyl, acarbobenzoxy, a propyl, a butyl, a pentyl, a hexyl, and a 3 to 20 carbonalkyl; and a second protecting group coupled to the carboxyl terminaland said carboxyl terminal protecting group is an amide.
 15. Thepolypeptide of claim 2, wherein said peptide comprises a firstprotecting group coupled to the amino terminus and a second protectinggroup coupled to the carboxyl terminus.
 16. The polypeptide of claim 2,wherein said peptide comprises an Ac group on the amino terminus. 17.The polypeptide of claim 2, wherein said peptide comprises an —NH₂ onthe carboxyl terminus.
 18. The polypeptide of claim 2, wherein saidpeptide comprises an Ac group on the amino terminus and an —NH₂ on thecarboxyl terminus.
 19. The polypeptide of claim 7, wherein said peptidecomprises an Ac group on the amino terminus.
 20. The polypeptide ofclaim 7, wherein said peptide comprises an —NH₂ on the carboxylterminus.
 21. The polypeptide of claim 7, wherein said peptide comprisesan Ac group on the amino terminus and an —NH₂ on the carboxyl terminus.22. The polypeptide of claim 2, wherein said peptide comprises a “D”amino acid.
 23. The polypeptide of claim 2, wherein said peptidecomprises a plurality of “D” amino acids.
 24. The polypeptide of claim2, wherein all enantiomeric amino acids comprising said peptide are “D”amino acids.
 25. The polypeptide of claim 2, wherein said peptide ismixed with a pharmacologically acceptable excipient.
 26. The polypeptideof claim 2, wherein said peptide is mixed with a pharmacologicallyacceptable excipient suitable for oral administration to a mammal. 27.The polypeptide of claim 6, wherein said oxidizing agent is selectedfrom the group consisting of hydrogen peroxide, 13(S)-HPODE,15(S)-HPETE, HPODE, HPETE, HODE, and HETE.
 28. The polypeptide of claim6, wherein said phospholipid is selected from the group consisting of1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC),1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC)),1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine (SAPE). 29.The polypeptide of claim 1, wherein said polypeptide is coupled to aphospholipid.
 30. The polypeptide of claim 29, wherein said polypeptideis covalently coupled to a phospholipid.
 31. The polypeptide of claim29, wherein said polypeptide is covalently coupled to a phospholipidcomprising lysophosphatidyl choline.
 32. The polypeptide of claim 29,wherein said polypeptide is covalently coupled to a phospholipidselected from the group consisting of propionoyl, butanoyl, pentanoyl,caproyl, heptanoyl, capryloyl, nonanoyl, capryl, undcanoyl, lauroyl,tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, heptadecanoyl,stearoyl, nonadecanoyl, arachidoyl, heniecosanoyl, behenoyl,trucisanoyl, lignoceroyl, myristoleoyl (9-cis), myristelaidoyl(9-trans), palmitoleoyl (9-cis), palmitelaidoyl (9-trans).
 33. Thepolypeptide of claim 32, wherein said polypeptide is covalently coupledto the sn-1 or sn-2 position of said phospholipid.
 34. A compositionsuitable for oral administration that ameliorates a symptom ofatherosclerosis, wherein said composition comprises a peptide comprisinga amphipathic helix having charged residues on the polar face of thepeptide and possessing a wide non-polar face, wherein said peptidecomprises a D amino acid and said peptide is blocked at the aminoterminus and the carboxyl terminus.
 35. The composition of claim 34,wherein said peptide is at least 10 amino acids in length.
 36. Thecomposition of claim 35, wherein said peptide is about 40 or fewerpeptides in length.
 37. The composition of claim 35, wherein saidpeptide comprises a G* amphipathic helix.
 38. The composition of claim37, wherein said peptide shows greater than about 50% sequence identitywith apo J.
 39. The composition of claim 35, wherein said peptideprotects a phospholipid against oxidation by an oxidizing agent.
 40. Thecomposition of claim 35, wherein said peptide comprises an amino acidsequence selected from the group consisting of LLEQLNEQFNWVSRLANLTEGE,(SEQ ID NO:1), LLEQLNEQFNWVSRLANL, (SEQ ID NO:2),NELQEMSNQGSKYVNKEIQNAVNGV, (SEQ ID NO:3), IQNAVNGVKQIKTLIEKTNEE, (SEQ IDNO:4), RKTLLSNLEEAKKKKEDALNETRESETKLKEL, (SEQ ID NO:5),PGVCNETMMALWEECK, (SEQ ID NO:6), PCLKQTCMKFYARVCR, (SEQ ID NO:7),ECKPCLKQTCMKFYARVCR, (SEQ ID NO:8), LVGRQLEEPL, (SEQ ID NO:9),MNGDRIDSLLEN, (SEQ ID NO:10), QQTHMLDVMQD, (SEQ ID NO:11),FSRASSIIDELFQD, (SEQ ID NO:12), PFLEMIHEAQQAMDI, (SEQ ID NO:13),PTEFIREGDDD, (SEQ ID NO:14), RMKDQCDKCREILSV, (SEQ ID NO:15),PSQAKLRRELDESLQVAERLTRKYNELLKSYQ, (SEQ ID NO:16),LLEQLNEQFNWVSRLANLTQGE, (SEQ ID NO:17), DQYYLRVTTVA, (SEQ ID NO:18),PSGVTEVVVKLFDS, (SEQ ID NO:19), PKFMETVAEKALQEYRKKHRE, (SEQ ID NO:20),WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO:21), VATVMWDYFSQLSNNAKEAVEHLQK(SEQ ID NO:22), RWELALGRFWDYLRWVQTLSEQVQEEL (SEQ ID NO:23),LSSQVTQELRALMDETMKELKELKAYKSELEEQLT (SEQ ID NO:24),ARLSKELQAAQARLGADMEDVCGRLV (SEQ ID NO:25), VRLASHLRKLRKRLLRDADDLQKRLA(SEQ ID NO:26), PLVEDMQRQWAGLVEKVQA (SEQ ID NO:27), MSTYTGIFTDQVLSVLK(SEQ ID NO:28), and LLSFMQGYMKHATKTAKDALSS (SEQ ID NO:29).
 41. Thecomposition of claim 35, wherein said first protecting group and saidsecond protecting group are independently selected from the groupconsisting of an acetyl, amide, 3 to 20 carbon alkyl groups, Fmoc,t-boc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group,9-florenecarboxylic group, 9-fluorenone-1-carboxylic group,benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt),4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),Mesitylene-2-sulphonyl (Mts), 4,4=-dimethoxybenzhydryl (Mbh),Tosyl(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl(MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl),Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom),t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum),t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), a benzoyl group, acarbobenzoxy group, a propyl group, a butyl group, a pentyl group, ahexyl group group, and Trifluoroacetyl (TFA).
 42. The composition ofclaim 35, wherein said first protecting group is an acetyl.
 43. Thecomposition of claim 35, wherein said second protecting group is anamide.
 44. The composition of claim 35, wherein more than half of theenantiomeric amino acids comprising said peptide are D amino acids. 45.The composition of claim 35, wherein all enantiomeric amino acidscomprising said peptide are D amino acids.
 46. The composition of claim35, wherein said composition further comprises a pharmaceuticallyacceptable excipient.
 47. The composition of claim 46, wherein saidexcipient is an excipient suitable for oral administration.
 48. Thecomposition of claim 46, wherein said excipient is an excipient suitablefor injection.
 49. A pharmaceutical composition, said compositioncomprising a polypeptide of claim 1 in a pharmaceutically acceptableexcipient.
 50. The composition of claim 49, wherein said compositioncomprises a unit dosage formulation.
 51. The composition of claim 35,wherein said polypeptide is coupled to a phospholipid.
 52. Thecomposition of claim 51, wherein said polypeptide is covalently coupledto a phospholipid.
 53. The composition of claim 51, wherein saidpolypeptide is covalently coupled to a phospholipid comprisinglysophosphatidyl choline.
 54. The composition of claim 51, wherein saidpolypeptide is covalently coupled to a phospholipid selected from thegroup consisting of propionoyl, butanoyl, pentanoyl, caproyl, heptanoyl,capryloyl, nonanoyl, capryl, undcanoyl, lauroyl, tridecanoyl, myristoyl,pentadecanoyl, palmitoyl, heptadecanoyl, stearoyl, nonadecanoyl,arachidoyl, heniecosanoyl, behenoyl, trucisanoyl, lignoceroyl,myristoleoyl (9-cis), myristelaidoyl (9-trans), palmitoleoyl (9-cis),palmitelaidoyl (9-trans).
 55. A method of ameliorating a symptom ofatherosclerosis in a mammal, said method comprising administering tosaid mammal a peptide or a concatamer of a peptide comprising anamphipathic helical peptide having charged residues on the polar face ofthe peptide and possessing a wide non-polar face.
 56. The method ofclaim 55, wherein said peptide is at least 10 amino acids in length. 57.The method of claim 56, wherein said peptide is about 40 or fewerpeptides in length.
 58. The method of claim 56, wherein said peptidecomprises a G* amphipathic helix.
 59. The method of claim 58, whereinsaid peptide shows greater than about 50% sequence identity with apo J.60. The method of claim 56, wherein said peptide protects a phospholipidagainst oxidation by an oxidizing agent.
 61. The method of claim 56,wherein said peptide comprises an amino acid sequence selected from thegroup consisting of LLEQLNEQFNWVSRLANLTEGE, (SEQ ID NO:1),LLEQLNEQFNWVSRLANL, (SEQ ID NO:2), NELQEMSNQGSKYVNKEIQNAVNGV, (SEQ IDNO:3), IQNAVNGVKQIKTLIEKTNEE, (SEQ ID NO:4),RKTLLSNLEEAKKKKEDALNETRESETKLKEL, (SEQ ID NO:5), PGVCNETMMALWEECK, (SEQID NO:6), PCLKQTCMKFYARVCR, (SEQ ID NO:7), ECKPCLKQTCMKFYARVCR, (SEQ IDNO:8), LVGRQLEEFL, (SEQ ID NO:9), MNGDRIDSLLEN, (SEQ ID NO:10),QQTHMLDVMQD, (SEQ ID NO:11), FSRASSIIDELFQD, (SEQ ID NO:12),PFLEMIHEAQQAMDI, (SEQ ID NO:13), PTEFIREGDDD, (SEQ ID NO:14),RMKDQCDKCREILSV, (SEQ ID NO:15), PSQAKLRRELDESLQVAERLTRKYNELLKSYQ, (SEQID NO:16), LLEQLNEQFNWVSRLANLTQGE, (SEQ ID NO:17), DQYYLRVTTVA, (SEQ IDNO:18), PSGVTEVVVKLFDS, (SEQ ID NO:19), PKFMETVAEKALQEYRKKHRE, (SEQ IDNO:20), WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO:21),VATVMWDYFSQLSNNAKEAVEHLQK (SEQ ID NO:22), RWELALGRFWDYLRWVQTLSEQVQEEL(SEQ ID NO:23), LSSQVTQELRALMDETMKELKELKAYKSELEEQLT (SEQ ID NO:24),ARLSKELQAAQARLGADMEDVCGRLV (SEQ ID NO:25), VRLASHLRKLRKRLLRDADDLQKRLA(SEQ ID NO:26), PLVEDMQRQWAGLVEKVQA (SEQ ID NO:27), MSTYTGIFTDQVLSVLK(SEQ ID NO:28), and LLSFMQGYMKHATKTAKDALSS (SEQ ID NO:29).
 62. Themethod of claim 61, wherein said peptide is a concatamer of two or moreof said amino acid sequences.
 63. The method of claim 56, wherein saidpeptide further comprises a protecting group.
 64. The method of claim56, wherein said peptide further comprises a protecting group coupled tothe amino or carboxyl terminus.
 65. The method of claim 63, wherein saidprotecting group is a protecting group selected from the groupconsisting of acetyl, amide, 3 to 20 carbon alkyl groups, Fmoc, t-boc,9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylicgroup, 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl(Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt),4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl(Mts), 4,4=-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl(MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom),t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum),t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), a benzoyl group, acarbobenzoxy group, a propyl group, a butyl group, a pentyl group, ahexyl group group, and Trifluoroacetyl (TFA).
 66. The method of claim63, wherein said peptide comprises a protecting group coupled to theamino terminal and said amino terminal protecting group is a protectinggroup selected from the group consisting of a benzoyl group, an acetyl,a propeonyl, a carbobenzoxy, a propyl, a butyl, a pentyl, a hexyl, and a3 to 20 carbon alkyl.
 67. The method of claim 63, wherein said peptidecomprises a protecting group coupled to the carboxyl terminal and saidcarboxyl terminal protecting group is an amide.
 68. The method of claim63, wherein said peptide further comprises: a first protecting groupcoupled to the amino terminus wherein said protecting group is aprotecting group selected from the group consisting of a benzoyl group,an acetyl, a propeonyl, a carbobenzoxy, a propyl, a butyl, a pentyl, ahexyl, and a 3 to 20 carbon alkyl; and a second protecting group coupledto the carboxyl terminal and said carboxyl terminal protecting group isan amide.
 69. The method of claim 56, wherein said peptide comprises afirst protecting group coupled to the amino terminus and a secondprotecting group coupled to the carboxyl terminus.
 70. The method ofclaim 56, wherein said peptide comprises an Ac group on the aminoterminus.
 71. The method of claim 56, wherein said peptide comprises an—NH₂ on the carboxyl terminus.
 72. The method of claim 56, wherein saidpeptide comprises an Ac group on the amino terminus and an —NH₂ on thecarboxyl terminus.
 73. The method of claim 61, wherein said peptidecomprises an Ac group on the amino terminus.
 74. The method of claim 61,wherein said peptide comprises an —NH₂ on the carboxyl terminus.
 75. Themethod of claim 61, wherein said peptide comprises an Ac group on theamino terminus and an —NH₂ on the carboxyl terminus.
 76. The method ofclaim 56, wherein said peptide comprises a “D” amino acid.
 77. Themethod of claim 56, wherein said peptide comprises a plurality of “D”amino acids.
 78. The method of claim 56, wherein all enantiomeric aminoacids comprising said peptide are “D” amino acids.
 79. The method ofclaim 56, wherein said polypeptide is coupled to a phospholipid.
 80. Themethod of claim 79, wherein said polypeptide is covalently coupled to aphospholipid.
 81. The method of claim 79, wherein said polypeptide iscovalently coupled to a phospholipid comprising lysophosphatidylcholine.
 82. The method of claim 79, wherein said polypeptide iscovalently coupled to a phospholipid selected from the group consistingof propionoyl, butanoyl, pentanoyl, caproyl, heptanoyl, capryloyl,nonanoyl, capryl, undcanoyl, lauroyl, tridecanoyl, myristoyl,pentadecanoyl, palmitoyl, heptadecanoyl, stearoyl, nonadecanoyl,arachidoyl, heniecosanoyl, behenoyl, trucisanoyl, lignoceroyl,myristoleoyl (9-cis), myristelaidoyl (9-trans), palmitoleoyl (9-cis),palmitelaidoyl (9-trans).
 83. The method of claim 56, wherein saidpeptide is mixed with a pharmacologically acceptable excipient.
 84. Themethod of claim 56, wherein said peptide is mixed with apharmacologically acceptable excipient suitable for oral administrationto a mammal.
 85. The method of claim 55, wherein said administeringcomprises orally administering said peptide.
 86. The method of claim 55,wherein said mammal is a mammal diagnosed as having one or more symptomsof atherosclerosis.
 87. The method of claim 55, wherein said organism isa mammal diagnosed as at risk for atherosclerosis.
 88. The method ofclaim 55, wherein said mammal is a human.
 89. The method of claim 55,wherein said mammal is non-human mammal.
 90. A method of ameliorating asymptom of a pathology characterized by an inflammatory response, saidmethod comprising administering to said mammal a peptide or a concatamerof a peptide comprising an amphipathic helical peptide having chargedresidues on the polar face of the peptide and possessing a widenon-polar face.
 91. The method of claim 90, wherein said peptide is atleast 10 amino acids in length.
 92. The method of claim 91, wherein saidpeptide is about 40 or fewer peptides in length.
 93. The method of claim91, wherein said peptide comprises a G* amphipathic helix.
 94. Themethod of claim 93, wherein said peptide shows greater than about 50%sequence identity with apo J.
 95. The method of claim 91, wherein saidpeptide protects a phospholipid against oxidation by an oxidizing agent.96. The method of claim 91, wherein said peptide comprises an amino acidsequence selected from the group consisting of LLEQLNEQFNWVSRLANLTEGE,(SEQ ID NO:1), LLEQLNEQFNWVSRLANL, (SEQ ID NO:2),NELQEMSNQGSKYVNKEIQNAVNGV, (SEQ ID NO:3), IQNAVNGVKQIKTLIEKTNEE, (SEQ IDNO:4), RKTLLSNLEEAKKKKEDALNETRESETKLKEL, (SEQ ID NO:5),PGVCNETMMALWEECK, (SEQ ID NO:6), PCLKQTCMKFYARVCR, (SEQ ID NO:7),ECKPCLKQTCMKFYARVCR, (SEQ ID NO:8), LVGRQLEEFL, (SEQ ID NO:9),MNGDRIDSLLEN, (SEQ ID NO:10), QQTHMLDVMQD, (SEQ ID NO:11),FSRASSIIDELFQD, (SEQ ID NO:12), PFLEMIHEAQQAMDI, (SEQ ID NO:13),PTEFIREGDDD, (SEQ ID NO:14), RMKDQCDKCREILSV, (SEQ ID NO:15),PSQAKLRRELDESLQVAERLTRKYNELLKSYQ, (SEQ ID NO:16),LLEQLNEQFNWVSRLANLTQGE, (SEQ ID NO:17), DQYYLRVTTVA, (SEQ ID NO:18),PSGVTEVVVKLFDS, (SEQ ID NO:19), PKFMETVAEKALQEYRKKHRE, (SEQ ID NO:20),WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO:21), VATVMWDYFSQLSNNAKEAVEHLQK(SEQ ID NO:22), RWELALGRFWDYLRWVQTLSEQVQEEL (SEQ ID NO:23),LSSQVTQELRALMDETMKELKELKAYKSELEEQLT (SEQ ID NO:24),ARLSKELQAAQARLGADMEDVCGRLV (SEQ ID NO:25), VRLASHLRKLRKRLLRDADDLQKRLA(SEQ ID NO:26), PLVEDMQRQWAGLVEKVQA (SEQ ID NO:27), MSTYTGIFTDQVLSVLK(SEQ ID NO:28), and LLSFMQGYMKHATKTAKDALSS (SEQ ID NO:29).
 97. Themethod of claim 91, wherein said organism is an organism diagnosed ashaving one or more symptoms of an inflammatory response.
 98. The methodof claim 91, wherein said organism is an organism diagnosed as at riskfor a pathology associated with an inflammatory response.
 99. The methodof claim 91, wherein said organism is a human.
 100. The method of claim91, wherein said organism is non-human mammal.
 101. A kit forameliorating a symptom of atherosclerosis, said kit comprising acontainer containing a polypeptide of any one of claims 1 through 28.102. The kit of claim 101, wherein said peptide is combined with apharmaceutically acceptable excipient.
 103. The kit of claim 101,wherein said peptide is combined with a pharmaceutically acceptableexcipient in a unit dosage formulation.
 104. The kit of claim 103,wherein said unit dosage formulation is for oral administration. 105.The kit of claim 101, further comprising instructional materialsteaching the use of said peptide for ameliorating one or more symptomsof atherosclerosis or of a pathology characterized by an inflammatoryresponse.
 106. A method of mitigating or preventing a coronarycomplication associated with an acute phase response to an inflammationin a mammal, wherein said coronary complication is a symptom ofatherosclerosis, said method comprising administering to a mammal havingsaid acute phase response, or at risk for said acute phase response, apolypeptide of any one of claims 1 through
 28. 107. The method of claim106, where said administration is by a route selected from the groupconsisting of oral administration, nasal administration, rectaladministration, intraperitoneal injection, and intravascular injection,subcutaneous injection, transcutaneous administration, and intramuscularinjection.
 108. The method of claim 106, wherein said polypeptide isadministered in combination with an all L-form of the same polypeptide.109. The method of claim 106, wherein said polypeptide is provided as aunit formulation in a pharmaceutically acceptable excipient.
 110. Themethod of claim 106, wherein said acute phase response is aninflammatory response associated with a recurrent inflammatory disease.111. The method of claim 107, wherein said acute phase response isassociated with a disease selected from the group consisting of leprosy,tuberculosis, systemic lupus erythematosus, polymyalgia rheumatica,polyarteritis nodosa, scleroderma, idiopathic pulmonary fibrosis,chronic obstructive pulmonary disease, Alzheimers Disease and AIDS,polymyalgia rheumatica, polyarteritis nodosa, scleroderma, idiopathicpulmonary fibrosis, chronic obstructive pulmonary disease, AlzheimersDisease, AIDS, coronary calcification, calcific aortic stenosis,osteoporosis, and rheumatoid arthritis.
 112. The method of claim 106,wherein said acute phase response is an inflammatory response associatedwith a condition selected from the group consisting of a bacterialinfection, a viral infection, a fungal infection, an organ transplant, awound, an implanted prosthesis, parasitic infection, sepsis, endotoxicshock syndrome, and biofilm formation.
 113. A method of mitigating orpreventing a coronary complication associated with an acute phaseresponse to an inflammation in a mammal, wherein said coronarycomplication is a symptom of atherosclerosis, said method comprising:assaying said mammal for an acute phase protein (APP) level indicativeof an acute phase response or a significant risk of an acute phaseresponse; and administering to a mammal showing an acute phase protein(APP) level indicative of an acute phase response a polypeptide of anyone of claims 1 through
 28. 114. The method of claim 113, wherein saidacute phase protein (APP) is a postive APR selected from the groupconsisting of serum amyloid A, c-reactive protein, serum amyloid Pcomponent, C2 complement protein, C3 complement protein, C4 complementprotein, C5 complement protein, C9 complement protein, B complementprotein, C1 inhibitor, C4 binding protein, fibrinogen, von Willebrandfactor, α1-antitrypsin, α1-antichymotrypsin, α2 antiplasmin, heparincofactor II, plasminogen activator inhibitor I, haptoglobin, haemopexin,ceruloplasmin, manganese superoxide dismutase, α1-acid glycoprotein,haeme oxygenase, mannose binding protein, leukocyte protein I,lipoprotein (a), and lipopolysaccharide binding protein.
 115. The methodof claim 113, wherein said acute phase protein (APP) is a negative APRselected from the group consisting of albumin, prealbumin, transferin,apoAI, apoAII, (α2-HS glycoprotein, inter-α-trypsin inhibitor, histidinerich glycoprotein.